Information

Why proteinase K doesn't degrade itself?


Can anyone tell me why proteinase K doesn't degrade itself?

If possible please provide me the source.


According to invitrogen proteinase K does undergo autolysis, but that some leftover fragments still have protease activity. The solution should be stored at -20C, this low temperature is probably the best way to prevent autolysis. If you're using the enzyme to digest protein at 37C, you'll just have to deal with losing the proteinase k during the reaction and make sure you add enough to destroy the target protein before the proteinase is depleted.


Proteinase K is sensitive for autolysis. The enzyme is stabilized by the presence of calcium ions (which bind to the protein). Absence of calcium promotes autolysis of the enzyme and also reduces the half life of the enzyme activity. The most likely reason why most researchers never notice an substancial effect of autolysis is that this takes around 48h to get noticed, while the most proteinase K digest take only a few hours. For details read the following paper:


Immunoprecipitation

Immunoprecipitation Notes

Immunoprecipitation (IP) is used to separate proteins that are bound to a specific antibody from the rest of a sample, while co-IP is used to identify protein–protein interactions between the protein that bound to the antibody used for IP and additional proteins that are detected by immunoblotting. It works by binding antibodies to Protein A, Protein G, or a lab-created mix of the two called Protein A/G. Proteins A and G are bacterial proteins that bind very well to antibodies. Proteins A and G bind differently to antibodies from different species, so you can look up a table to identify which protein will work better for you, or you can just order the Protein A/G (it’s not much more expensive, if at all). These proteins are bound to agarose beads (or patented variants) to give them weight. The basic idea is that you incubate your IP antibody with the agarose beads conjugated to Protein A/G (some labs call these beads “bugs”) and then incubate your sample protein solution with the antibody–agarose bead complex. You then centrifuge the sample at low speed, and the beads–antibody–bound protein complex will form a pellet, which you can separate from the supernatant and resuspend in another solution. Congratulations, you have performed an IP. If you want to perform co-IP, you take your IP product (collecting proteins in a very gentle lysis buffer to preserve protein complexes), run in SDS-PAGE, and perform immunoblots with antibodies that target proteins you think interact with your IP product protein(s). IP controls are similar to immunoblot controls, except that it really helps to have two antibodies that share the same target, so that you can immmunoprecipitate with one, and probe an immunoblot with both (if the IP was true, then both antibodies will stain the same bands). Again, the Abcam Protocol book is a great resource, as is the Thermo Scientific Tech Tip #64. Note that co-IP is different from Far-Western blotting, in that Far-Western blotting is examining protein–protein interactions (like co-IP) but uses a labeled “bait” protein to pull down interacting proteins, instead of using antibodies.

One should remember that IP, immunoblotting, and immunohistochemistry are considered three different techniques for a reason. IP has different experimental conditions than immunoblotting, both of these are different from immunohistochemistry, and those three are different from ELISAs (not covered in this book) basically, some antibodies work for one or more applications, and an antibody that works for all applications is exceedingly rare (I’m not sure one exists!). Further, IPs depend on both the ability of your primary antibody to bind the correct protein and the antibody’s ability to bind Protein A/G, so if one of those two things doesn’t happen, you won’t get data.


Questions

Q: Why don't you stop the Proteinase K reaction with glycine?
A: I think glycine inhibits the enzyme and stops the digestion immediately. I'm not sure when or why that step was removed from the protocol I'm using, but the repeated washes in PBT accomplishes the same thing more gradually. In the various protocols I've seen, people use a very wide range of ProtK final concentrations (from 4-50 µg/ml) and incubation times (1-10 minutes). In general, it seems like higher concentration goes with shorter incubation time and the use of a glycine buffer to stop the reaction: if the reaction time is short, the margin of error is smaller and you will have more control over stopping the digestion at the precise time by using the glycine. With lower concentration and longer incubation time, the timing of stopping the digestion is not so critical and the gradual removal of the enzyme with repeated washes is OK.

Q: About the Proteinase K: different manufacturers produce ProtK that differ in the amount of units of activity/mg. In the past, I used units/ml, rather than mg/ml. Did you ever pay attention to that, or do you think it is not reliable?
A: It doesn't matter what you use, units or mg per ml, as long as you know what the activity is for this application. Their 'units' don't measure digestion activity on fixed Drosophila embryos and you must discover the optimum performance conditions of your own stock of ProtK through testing. On the other hand, those units will give you a good estimate on where to start in the titration of your own stock if you are moving between brands or making up a fresh batch. One last comment about using ProtK: it definitely increases the absolute signal level of your stains, as well as reduces the background. It's worth the extra trouble.

Q: Why don't you use PCR to generate your probe templates? That way you don't need to grow up a lot of plasmid DNA.
A: Because I'm hopelessly old-fashioned and don't really know how to do PCR. Roche offers a PCR protocol for making labeled probes, which many people use, and the BDGP gene expression project uses a PCR protocol for its high-throughput embryo staining. I'm doing Qiagen midi-preps to purify good amounts of clean cDNA plasmids. However you like to make probe, do some quality control, such as running the reaction product on a gel, to make sure that the reaction went well, or detecting a spot of it on a membrane, to check the degree of labeling.

Q: Why don't you use DEPC-treated ddH2O? It's cheap and easy to make up.
A: I don't use it for any part of the embryo staining because I have no problem getting a lot of signal and therefore assume I've got no significant degradation of the probes after they are hybridized to the embryos. If you have an RNase contamination problem in your ddH2O water supply, then, by all means, you must fight back. Also, I prefer the commonly available non-DEPC treated, filtered, certified nuclease-free ddH2O in the probe synthesis reactions because I've heard even the smallest amount of residual DEPC can have a negative impact on the polymerase. Superstition? Probably, but 1 L of fancy RNase-free water, enough to synthesize about 50,000 probes, costs the same as 5 ml of DEPC and also you don't have to treat the ddH2O yourself.

Q: Why do you fragment your probes? I've heard you don't have to do this, and actually makes probes worse in some cases.
A: I don't claim any authority on this point, but I know what I've tried and the results I got. I am trying some experiments in which full-length, non-hydrolyzed probes are required, and find that they give terrible background problems. The exact same probes hydrolyzed give no background. I'm starting to get the impression that somewhere between 0.5-1 kb probe size, fragmentation becomes necessary. So some of the differing opinions on this point, to fragment or not to fragment, could be due to experiences with different probe sizes. I've heard also that there is an optimum average probe size to detect EACH gene's transcript. Why not?

Q: Sometimes after the hybridization steps I get big clumps of embryos that never break up. Why?
A: I'm pretty sure it's due to excessive Proteinase K treatment. Someone else once asked me about this problem, and apparently after modifying his ProtK digestion conditions, the problem was gone. I have no idea why this clumping occurs, and it is truly horrifying to see it happen to precious embryos.

Q: That is a very long hybridization step, I normally do only 12-18 hours. Are you sure the quality of the in situ increases so much with those extra hours?
A: In 50% formamide it takes probes a very long time to come to equilibrium with their proper targets, while being prevented from forming stable hybrids with their mismatch targets. Several people I have talked to about this have confirmed that longer hybridization times have dramatically improved the performance of probes that before gave weak stains. In these cases, the hybridization time was extended from one overnight (

36-40 hours). I saw one reference where they went up to three overnights trying to detect very unabundant transcripts! I have not tested a time series myself. Currently, my embryos are coming out with very good signal at 20-24 hours, and also with very good morphology. I'm not sure, but it seems like the two overnight hybridization makes them softer and more prone to become warped on the slide. After all, we're baking a proteinaceous tissue structure for a long time in a strong denaturant.

Q: What about your 55° C. hybridization temperature? Why so low? I'm doing in situs at 65° C.
A: The original whole mount non-radioactive in situ hybridization technique, described in 1989 by Tautz and Pfeifle, employed labeled DNA probes and hybridization conditions that very closely resemble those used in the 1980's for DNA:DNA filter hybridizations: 50% formamide and 45° C. In the embryo, DNA:RNA hybrids were formed and those conditions worked. Shortly thereafter, people started using RNA probes, and the rule of thumb, that for a given sequence, the melting temperature of the RNA:RNA hybrid was 10° C. higher than the corresponding DNA:DNA hybrid, was probably used to establish the 55° C. temperature for RNA probes. I don't know exactly why or when people started using higher temperatures (60° C. and higher), but my suspicion is that it was in response to having problems with unacceptably high background staining caused by non-specific sticking of probes to the embryos. I've been hybridizing embryos at 55° C. for a long time without a problem, and the background problems I've encountered attempting these fluorescent methods have been due solely to the primary detection reagents, not the probes, according to my controls.


SOJ Biochemistry

Microbial Ghosts (MGs) is a new term that describes evacuated and dead microbes. Apparently, MGs will be able (soon) to substitute another term the "bacterial ghosts (BGs)". A new protocol for preparing MGs was introduced using the critical concentration or amounts of some chemical compounds and enzymes. In principle any compound such as SDS and NaOH or enzyme such as lysozyme and Proteinase K that could induce a pore(s) in the microbial cell wall could be used. "Evacology" might be a name for a new science that deals with living cells and viruses' evacuation. In addition, the biocritical concentration of H2O2 enables Virus Ghosts (VGs) preparation. The bacteriophage E lysis gene based protocol is restricted only to the gram-negative bacteria. The Sponge-like Protocol (SL) has opened the window to nearly all microbes and all biological cells and viruses to be prepared as ghosts. In this shift point this review aims to cover the most important information about such a topic. SL protocol is based on determining the critical concentration of compounds that can kill, make pore(s), evacuate the cells, but did not deform or affect the cell wall or their antigens (under such concentration). Lysozyme has been used in the original protocol to complement any deficiency result in survive of any of the E. coli cells. Lysozymes and Proteinas K can stand-alone or can be combined with the other possible chemical compounds. The SL protocol for ghosts preparation is simple, inexpensive, in house, reliable, safe and cause pores starting from outside the cells to their inside. The future will show rising interest with such simple protocol, which could allow us to prepare our vaccine and drug delivery different ghosts' related formula in kitchen. In this, review most of the experiences gained from practicing experiments in ghosts' preparation and some idea about such subject were summarized and discussed.

Keywords: Bacterial Ghosts Microbial Ghosts Biological Ghosts Sponge-Like protocol Bacteriophage E lysis gene

Like any creature, each microbe has a life cycle, which end with death. The microbes' cell walls after their death could stay longer and resist decaying [4-7]. However, due to the process of the natural decay, environmental effect, enzymatic activity of other microbes, or any other expected mechanism for a pore or pores formation, natural loss of the cytoplasm or in better words, natural microbial ghosting will be happened. As being parts of the nature, microbes after being dead they are affected by their surrounding ecological environment. For that, cell ghosts and microbial ghosts are produced daily in our bodies (inside, or outside). They are produced in the lung, in the stomach, in the surface of our skin, in our aged food and so on. Therefore, dead and ghost microbes are natural phenomena.

In addition, they play different roles in immunization. Natural Ghosting phenomenon in our bodies plays important roles in immunization. Aged foods used by old civilization might have been used for the aim of immunization. The ancient Egyptian invented some type of cheese which named old cheese or Mish (Mesh) which combine between being so old so if any harm microbe is existed as a contaminate it is either being attenuated or dead. Such old cheese or Mish/Mesh cheese might be invented not as a type of cheese only but to protect the Egyptian from many diseases.

The ϕX174 E lysis protein cause E specific transmembrane tunnel structure built through the cell envelope complex [24-27]. It form a fusion through the inner and outer cell membranes, forming a specific transmembrane tunnel structure. Through such tunnel or pore, the bacterial cytoplasmic content is passing out. Genetic engineering and molecular biology tools enables better control for the bacteriophage E lysis gene based protocol.

The produced lysis enzyme enable pores in the bacterial cell wall. There is still a need for degrading the residue of the DNA, plasmid, lysozymes, proteinase K and so on, which are still contaminating the opened bacterial cells.

E lysis gene, its activity is restricted only to the gram-negative bacteria. The gene E was cloned and expressed in different gramnegative hosts. Such expression has been controlled by a heat sensitive promotor, which allows the expression of the E lysis gene. Using the heat sensitive promotor and regulator were used for better control. Nevertheless, its main weak point that it is restricted only to the gram-negative bacteria [4, 28].

Gene E: The amino acids content of the E lysis protein sequence of the Enterobacteria phage ϕX174 and its nucleotides content were well identified (as below). One can obtain uncut phage DNA from the market and clone the E lysis gene. Alternatively, one can isolate the phage and made DNA isolation then clone the E lysis gene or cut it with suitable enzyme and made gene library.

E lysis gene nucleotides sequence:
atggtacgctggactttgtgggataccctcgctttcctgctcctgttgagtttattgctgccgtcattgcttattatgttcatcccgtcaacattcaaacggc ctgtctcatcatggaaggcgctgaatttacggaaaacattattaatggcgtcgagcgtccagttaaagccgctgaattgttcgcgcttaccttgcgtgt acgcgcaggaaacactgacgttcttgctgacgcagaagaaaacgtgcgtcaaaaattgcgtgcagaaggagtga

Amino acids sequence:
MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNLRKTLLMASSVQLKPLNCSRLPCVYAQET LTFLLTQKKTCVKNCVQKE

One pore is enough!: A pore formation in the microbes' cell walls will lead to the removal of the cytoplasm which come out due to the cell wall imbalanced pressure force, osmosis differences, mechanical pressure, etc. The external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid BGs [27]. Apparently, and after observing many of the electron microscope images only one pore is usually existed. That might be due to the force happened as the result of the existence of the first pore. When first pore is being opened it imbalance the internal pressure lead to getting rid of the cytoplasm contents and the rest of the E lysis protein so there is no chance to form another pore [29].

Foreign surface antigen and drug delivery: Ghost cells can be performed after the expression of foreign antigen, loading drugs, DNA, plasmid etc to the cells. However, in case of using MGs in the drug delivery, after such loading to the drug, the existed pore must be closed or in better word must be sealed [30]. Recently, another tactic have been introduced where Saccharo-myces cerevisiae cell ghosts were loaded with dissolved ghosts gossypol acetic acid, then the dissolved ghosts gossypol acetic was allowed to crystallize inside the yeast cells. That will give the chance to load drugs without sealing the cells.

SL protocol introduce the idea of using the critical concentration of chemical compounds and recently enzymes for the ghosts preparation. The used steps were selected preciously to do in sequence steps enable full evacuation for the treated microbes or cells from their cytoplasmic contents. The future might show a more perfect chemical compounds or a strong modification in the protocol but the Acknowledge should be given to the original six used chemical compounds. They are, NaOH, SDS, CaCO3, H2O2, NaCl and Ethanol. Recently NaHCO3 was used instead of CaCO3 to produce yeast ghosts. H2O2 has stood alone to degrade Newcastle RNA using bio-critical concentration. Enzymes, which can affect on the microbial cell wall such as lysozyme and protinase k are under optimization to give the equal results of the SL protocol [data not shown] but chemical compounds are more cost effective and omit the risk of incorrect immunization due to the use of enzymes which are protein in their nature [31]

The main idea of the SL protocol: The SL protocol main idea is simple and applicable it depend on determining the (MIC) Minimum Inhibition Concentration and the (MGC) Minimum Growth Concentration of the used compounds or enzymes. The minimum killing effect in case of using MIC of the used compounds or enzymes they should cause minimum effect on the dead cells. In case of MGC the cells still alive. However, by using another MIC/ MGC for the another compound(s) plus the physical effect of the repeated centrifugation steps, that all will lead to empty dead cells but with correct 3D structure and correct surface antigens which enables correct immunization upon the use of the experimental animals. SL protocol gives the chance to prepare ghosts from gram-positive and gram-negative bacteria, yeast, and virus and so on. Such a concept could be pass the microbes to any other biological cell forms. Many other forms from the biological system will join the ghost family after their preparation by this protocol (critical chemical concentration) soon.

Using critical chemical concentration for microbial killing is a natural phenomenon: P. aeruginosa is a dominant microbe in the hospitals while it is – in one word - hydrocarbon biodegradable microbes. H2O2 could turn any of the P. aeruginosa to mucoid strain hence to alginate producer. P. aeruginosa is able to be mutated and to produce a huge amount of exopolysaccharide, mainly the alginate if exposed to the H2O2. That also could be happened in the patients' lungs. As a defense mechanism against the P. aeruginosa infection the lung produces H2O2. How could the lung cells adjust the amount of the H2O2 to kill the P. aeruginosa but not to kill their own cells? In general, the lung cell must produce (somehow) H2O2 in critical concentration to kill such microbes and not to damage or to kill its own cells [32]. For one or another purpose, some of the P. aeruginosa cells were not killed by the killing dosage of the H2O2, but were exposed instead to less amounts of the H2O2 by one or another mechanism. Less amount of H2O2 will induce mucoid mutant. Such mutants are able to produce a huge amount of exopolysacharid. From such natural phenomena one can understand some facts about our "biological system" and how it could use the concept of "the critical chemical concentration" intelligently. That includes:

1. Our biological system knows how to produce chemical compounds in critical concentrations.

2. Concentrations less than the MIC could cause mutation, where such concentrations could keep the pathogens affected but alive.

3. Compounds produced by our biological system such as the oxidants and free radicals particularly those which produce to control pathogens should be given more concerns.

4. Misused of antioxidants could deteriorate our endogenous oxidant defense mechanism. For that, antioxidants should be taken wisely.

5. The biological system still proves that it is designed in perfect and intelligent way, so mechanisms used by such system should be given priority.

6. Lysozymes, proteases, DNases and other natural enzymes which are part of our defense system should be given more concerns to find more intelligent mechanism to control such pathogens.

7. Potant compound could be used upon dilution to do minimum side effect.

El-Baky and Amara(2014) combine between such phenomena and the idea of the SL protocol to in vitro degrade the Newcastle virus RNA to turn it to ghosts, which might be optimized to be in vivo protocol for controlling some pathogens without harming our biological system [3].

MGs omit the most important virulence factor: When foreign microbe enters to our body our immune system starts to react with it and the battle will base on their number, type and our immune system quality. However, some microbes have extra virulence factors, which could be collectively stronger than our immune system. So, even, we are strong enough, the microbe can be fatal and exceed the speed of our immune system. Such conditions cause death, or severe illness or disorder. For that, scientists prepare killed microbes or attenuated ones to be sure that the immune system will manage the situation and control the invasion. Such in / less-active microbe(s) give our immune system the chance and the time to react with it. However, there are many reports prove that attenuated microbes can be turned to virulence ones in some conditions.

For that scientists spent time to reduce the virulence factors of the pathogenic strains or to find alternative solutions. Such alternative solutions might be summarized as follow:

1. Repeated cultivation, in media did not maintain the microbe's virulence factors.
2. Cell aging.
3. Viruses can be grown in an unspecific host, which induce safer mutants.
4. The use of recombinant strains enable expressing antigen(s) of some pathogenic microbes on their cell wall surfaces.
5. Genetically modified pathogens with less or completely deactivated virulence factors can be used.
6. Safe strains could induce antibodies which can protect against some virulence pathogens.
7. Close species are used which are able to induce immunization against certain targeted pathogens.
8. Using MGs (Figure 3).
9. Totally killed microbes but with with a suitable of effective surface antigens (Some killing process affect severely on the microbes surface antigen).
After that, our immune system produces the suitable antibodies. And, become ready for any pathogenic invader.

MGs are truly dead cells and they could not replicate. For that, if they are pathogens they will lose the chance to win the battle against our immune system (by increasing their numbers). For that, they are perfect candidates for activating safely our immune system. Old civilizations have aware by the knowledge and the tools of the vaccination. They know how to vaccine against the smallpox, the disease which kill millions of peoples. They use unsuccessful virus legion of smallpox to vaccine-uninfected patients. The original name of the vaccination is variolation or inoculation. Even such reports prove that the main idea was transferred to the new world, only after doing some improvement in a method from the Middle East and Africa, which was known as ventilation. We still -until nowadays- use the same old technique. Such simple tools saved the life of unknown number of peoples. It is an African practice is transferred to America nearly in 1706- 1721 by a Sudanese slave [33]. However, it might be a shift in the concept when cowpox was used instead of the smallpox to give the full immunization against the later.

Some concerns about our immune system: There are some points that should be considered for those who are involved in the battle of the pathogens control. Some of such important concerns

concerns can be summarized as follows:

1. Our immune system is active in certain ages and less active in another. For that, we should be vaccinated in the correct time.

2. However, in case of using dead cells like BGs the equation is different. The vaccine types and the rout of administration is an important issue. Weak immune system, should give a correct time, formula (dead/attenuated microbes), site of administration and so on. MGs even an empty dead cells but it still have the correct 3D structure, so the immune system react with it with nearly the same power when it attack living cells. But one should consider the correct used number of cells. For that better and safer immunization were reported in case of using the MGs particularly for immunization.

3. One could take the maximum dosage from MGs safely while they are dead cells.

4. The genetic engineering and the molecular biology tools enable introducing or expressing the protein of the fatal microbes in the surface of recombinant safe microbe(s). After that the microbes turned to ghosts and become dead and safe.

5. BGs can be given safely in newly root of administration such nasal or on the surface of the wounded skin.

6. As a perfect biological package targeted by different immunological cells particularly the macrophage, they become the best choice for the gene therapy. BGS have increased our understanding to some biological factors such as our understanding to the resistance where intake cells (but not dead) might acquire new genetic material made them survive. Alternatively, before they loses their content under certain condition they might hybrid with other MGs.

7. Exopolysaccharide microbes could give false MIC where some microbes are survived due to biofilm formation. For that, microbes, which can be affected or mutated by the used chemical compounds, are recommended to be killed by NaOH using its MIC concentration first H2O2, SDS can induce exopolysacchaide production. As the other empty similar containers, MGs have more respective size and stable envelope and can be used in the field of the drug delivery.

8. BGs can be used in the diagnostic Kit as a reference antigen, where it should give positive reaction with serum containing the proper antibodies [34].

9. BGs will be in its better form after removing their cytoplasmic contents including the DNA, RNA, and the protein and so on.

The protocol can use either the introduced chemical compounds or enzymes in critical amounts or the combination between both. The original SL protocol and its reduced and modified forms were succeeding to prepare BGs from gram-negative, Gram-positive, yeast and viruses till nowadays. In fact, the concept of the protocol enables preparing ghosts from any microbe or even from any biological cells. That because it based on some Figure 3: The unique criteria for M. chemical compounds or enzymes were selected based on their ability to kill the microbes and induce pores in their cell walls, degrade/or remove their DNA, RNA and the protein. The compounds were used in concentrations that enable induction of minimal effect on the cell wall and the protocol itself use physical parameters to get rid of the cytoplasm such as the shaking and the cell pressing using centrifugation. For that, it is given the name SL protocol. The lysozyme and proteins K which are able to lysis most types of different microbes will give another chances for improving a commercial protocol for ghosts preparation. In fact, most of the molecular biologist have used such enzymes to prepare the DNA from various microbes. Only, they are in need to be used in critical activities enables ghosts preparation rather than the lysis of the microbes.

The first used chemical compounds: The selected compounds, which were used to prepare BGs using SL protocol, are NaOH, SDS, CaCO3, and H2O2 and both of NaCl and Ethanol are included. Lysozyme and proteinase K upon their use in a critical amount also give the same results. Some physical factors are involved (Centrifugation, shaking and temperature). Biological parameters are playing the central role in the success of the protocol such as, the type of the used cell or the microbe and their age. The ghosts' preparation condition(s) are effective factor(s). Such as cells density during the preparation. The cells quality should be monitored either by light or electron microscopes. The ability of the prepared ghosts' cells to induce correct immunization should be investigated. The sequence of the treatment is also an important issue. For example, exopolysaccharide-producing microbes must be treated with NaOH firstly. After preparing MGs, the cells must be investigated for the existence of any viable colonies. In addition, the viable cells should be deactivated, converted again to ghosts or the overall batches should be sterilized (in case of fatal microbes). It is might be interesting to highlight that the vaccine and the immunological technologies are fine technology and in some cases grams from correctly prepared MGs is in need to satisfy the demand.

Why NaOH and Why SDS? : NaOH and SDS are two wellknown compounds used for plasmid isolation from gram-negative bacteria. The protocol is given the name "alkaline lysis protocol for plasmid isolation". The lysis buffer made from ten percentage NaOH (not autoclave) and SDS (need to be autoclaved) as stock solutions (400 μl of the ten percentage SDS on 3400 μl water and then add 80 μl of ten percent NaOH). Both of NaOH and SDS are used to prepare the lysis buffer by mixing them with water. SDS introduces pores in the bacterial cells. For that, it is added to most of the toothpaste, used in lysis buffer for the plasmid isolation and for some of the DNA isolation protocol. If one use SDS in its MIC that might give minimum effect and introduce pores in the bacterial cells, such concept was one of the successful keys to isolate the DNA from any gram-negative microbes without a need for buying expensive kits. Increasing the temperature and the exposure time were additional option in case of gram-positive bacteria [35]. NaOH prove to be effective on the cell wall and for that, it is better to use its MGC. However, it is a potent bacterial killer. For that, it is recommended to use it at the first to deactivate strains able to be mutate and to resist the ghost preparation steps.

Why MIC and MGC?: MIC is a critical point where the used chemical, drug, enzymes or the antimicrobial agents are able to kill the microbe under the investigation condition with minimum side effect on their cells. In the serial dilution experiment, the tube after the MIC, which shows the first growth and which, given the name MGC such concentration should still effective on the microbe somehow. Using the concept that chemical compounds and enzymes were used in their MIC in a time or a combination between the MIC and MGC of other compounds this will support the concept of generating the minimum effect on the cell wall of the microbial strains. In another word, the used combinations collaborate to do relaxed job by complementing each other.

Experimental Design-Plackett-Burman, Box-Behnken and the excel solver for optimization: Experimental design has been used to optimize many of the biological processes. It has been also used to optimize the production of the BGs. Experimental design needs two levels for each variable, one is low and the other is high. And, it is preferable to use from four to ten variables. In the original protocol twelve experiments are conducted. Each experiment contains either the high level or the low level of each of the used microbe. All of the used experiments must be different and followed the Plackett-Burman design. Therefore, concerning the used chemical compounds concentration the MIC is the higher one +1 and the MGC is the lowest one -1 [36].

CaCO3 or NaHCO3: CaCl2 is used in the competent cells preparation. CaCl2 is able to facilitate the movement of the plasmid from outside the cells toward its inside. It can do the same with the SDS and other compounds.

Using another stronger "Ca" based compound will give better results with some microbes. Some could have dual activities as a membrane transfer and as an alkali such as CaCO3 and NaHCO3. CaCO3 was selected because it has another unique property that it is poorly water dissolved even after its autoclavation and it can be used as a suspension.

NaHCO3 was used with the first Eukaryotic prepared as ghosts using the idea of the critical chemical concentration [37,38].

Ethanol: Ethanol can be used to precipitate the DNA and protein if used as 90 percent. If it is used as 70-percent concentration that enables both of precipitation and salts elimination, (The 30 percent water content enables that). However, if it is used in concentration less than 70 percent it could eliminate the salt, DNA, RNA and the soluble protein as well. In the SL protocol, 60 percent of the ethanol was used to evacuate the bacterial cells content from their DNA, RNA and the salts.

Lysozyme: Lysozyme is an enzyme found in some tissues and secretions and is considered as a part of the defense mechanism against pathogens while it is able to lyse some microbes. The mechanism of its activity is mainly by its targeting to the bacterial exopolysaccharide existed in the cell wall causing osmotic shock or lysis. The most famous source is the hen egg, saliva, milk and bacteriophage T4. Its substrate is consisting of alternate residues of (1-4)-linked β-N-acetylemuramic acid (MurNAc) and β-N-acetylelucoseamine (GlcNAc). Lysozyme hydrolysis the bond between C-1 of β-N-acetylemuramic acid and C-4 of GlcNAc. Chitin (β-1-4-linked GlcNAc) is also a substrate [31].

Proteinase K: One of the endopeptidases able to digest keratin and it is a broad-spectrum serine proteinase. It is activated by calcium. It is used to remove protein contaminating nucleic acid. The enzyme's activity is stimulated by denaturants such as SDS. It is always used side by side with lysozymes. Proteinase K can be used to improve the MGs preparation [39].

Centrifugation: What can be happened if the pore in the microbial cell wall is so small and the bacterial cells still able to maintain their content (even the microbe have such a small pore). For solving such a problem, centrifugation will be the best process for pressing the microbial cells.

After introducing single pore (small or big) or more than single pore the microbe cells might still be able to maintain. Their cytoplasmic content due to the preplasmic membrane, the centrifugation will be able to press the cells to get rid of their contents. One can imagine the cells coming down to the test tube bottom due to the centrifugation force. After being settled and aggregated. The cells pressed like the sponge and the cells continue to get rid of their contents. Therefore, the protocol has been given the name SL protocol. So one should use a suitable centrifugation speed. One should not exceed 4000 rpm/min speed during the ghosts preparation. Speed from 2000 to 3500 rpm/min will give better cell quality.

Washing: Washing is an important step that because after the cultivation and during the centrifugation step the microbe surface and biomass trap debris and fatty acids. Such cells contaminating elements should be eliminated. In most cases, such cytoplasmic constituent or the rest of the growth condition could neutralize the effect of the used chemical compounds or enzymes. Also, washing enable getting rid from the elaborated cytoplasmic contents. So several washing steps using saline solution could improve the MGs quality. In fact after each centrifugation step samples from each supernatant should be investigated spectrophotometerically or by using gel electrophoresis to monitor the remove of the DNA and the protein. After the success of the protocol, its steps can be reduced and optimized.

The BGs Quality: The above step is concerned with monitoring the remove of cytoplasm which is not an indication about that the microbial cell walls are safe and did not deteriorate or damage. One question is immerged, how to evaluate the quality of the MGs cell walls during the ghosts preparation steps? The quality of the prepared MGs is an important issue. Light microscope should give correct judgment. However, electron microscope will give sharp evaluation for the bacterial 3D structure. To evaluate the quality of the BGs one should count randomly in a certain area ten bacteria and count the number of the cells which have correct 3D structure and use them as a percentage. Hemocytometer can be used for more precise results. Both of the scanning and transmission electron microscopes can be used to evaluate the quality of the MGs.

Electron microscope is absolutely proof that the prepared BGs or MGs are excellent, very good, good, or damaged cells. In case of using the electron microscopes, simply one can spread one drop of distilled water on the surface of the microscope slide. After that the diluted MGs have been added, spread gently and left for air dry. After being well dried one should rewash the microbial smear to remove any of the salt crystals (to get better electron microscope result) [40]. The microbial smear then dried again. In case of transmission microscope the standard protocol is used.

1. Prepare a pure and an identified microbial strain, simply by doing streak method for spreading the cells in the proper medium or on the selective medium plate. You should be care that the used medium does not mutate your strain.

2. Pick up single colony and re-spreads it on the surface of suitable medium and remark its phenotype.

3. Test the bacterial cells using light microscope for being sure of its type and purity. Both of simple stain and gram stain should be used.

4. Pick up a single pure colony either by sterile needle or by tooth pick and inoculate it to 25 ml flask containing 10 ml of the proper broth medium. And incubate for overnight. Fresh cells will give better MIC and MGC result.

Determination of the MIC and MGC for NaOH, SDS, and H2O2, enzymes etc.

1. Prepare several test tubes containing 5 ml or 4.5 ml of the proper medium aiming to conduct the serial dilution experiment.

2. Add 0.5 ml from the above solutions each in the first test tube of a set of tubes (7 tubes) and transfer 0.5 ml from one to second tube as in the standard protocol of the serial dilution. Remove 20 μl of each tube and add 20 μl of about 10 8 of the overnight cell culture in each tube (for each of the above chemical compound related experiment). Any change in the dilution should be considered. Incubate the tubes at 37°C for overnight (or at the growth temperature, which is recommended for the microbe under investigation). Calculate the MIC and the MGC for each of the used chemical compounds.

3. In case of CaCO3 it was used either as 1.05 μg/mL or 0.35 μg/mL was used.

After collecting, the aged cells by centrifugation and washing them with saline several times upon the type of the used microbe one can follow one of the below strategies.

Strategy No. 1 (The original SL protocol with twelve Plackett- Burman experiments protocol) (Figure 4).

The original used method is based on using 5x concentration strategy. NaOH, SDS and CaCO3 were used in one step. Then the washed cells treated with H2O2 in the second step. After enough washing and centrifugation steps, 60 percent ethanol is used in the third step. Each of the twelve used experiments is following the Plackett-Burmen design as in the original protocol [41].

Strategy No. 2 (The Plackett-Burman reduced protocol) (Figure 5) same as in the strategy no-1., exactly but only the best two experiments were selected. Such reduced protocol was designed to be used only for the similar strains such as E. coli BL21 and JM109. However, it has been used for other microbes and proved to be effective [42].

Strategy No. 3 (The 2x strategy) (Figure 6), one can use 2x concentration of the used chemical compounds each in separate step instead of 5 x. For example add one ml of the suspended bacterial cells to one ml of 2x of the NaOH (NaOH 10 percentage is sterile by itself but if diluted one should avoide any contamination). The 2x stock (prepared according to the results of the MIC and the MGC as recommended, in Plackett-Burman reduced protocol) are prepared and added to the bacteria. That depends on the experiment one or two in the reduced protocol. After finishing the treatment, centrifuging the cells and washing, the treatment with the 2x of each compound of MIC/MGC is started. That enables us to use NaOH alone with the expolysaccharide producing microbe to kill it and to take from the cells the chance to pro-duce the exopolysaccharide.

Strategy No. 4, (The special protocol for mutated strains such as exopolysaccharide producing strain ( for example P. aeruginosa). After working with an expolysaccharide producing strains, or eukaryotic and viruses strains. It becomes clear that H2O2 might induce resistance. In such case, NaOH must be used in the first treatment to kill the microbial cells and to prevent it from producing the exopolysaccharide. And in the same case the MIC must be test correctly while exopolysaccharide will give wrong result. In case of obtaining wrong MIC one can use the MIC which which is calculated the E. coli or use lesser concentration while exopolysaccharide will give high MIC value [43].

Strategy No. 5 Using enzymes after determining their MIC and MGC side by side with the used chemical compounds particularly with some gram-positive and halophilic strains [under optimization].

The SL protocol is a simple, inexpensive and in-house protocol. However, one aim of this review is to give the reader the most tested tactic, problems, solutions and image during his experiment design. This review should be read in detailed steps to enable the reader to implement or design his own protocol for a particular microbial strain. The protocol can be used to prepare ghosts from nearly all the bacterial strains, yeasts, and viruses. One can use any of the strategies explained in this protocol and included in this review. It is important to determine the MIC of the used chemical compounds and enzymes to start the protocol. One should also know some important information about the used microbes either gram-negative or positive, exopolysaccharide producer, the strains ability to be mutagenized and so on.

The following advices will give the reader some image during his experiment design. This review should be read carefully to gain all the idea within.

1. You should do all your experiment under aseptic condition and well-planned microbiological experiment in a microbiological lab containing all the needed facility. For example any contamination with a spore former microbe such as Bacillus sp will give wrong MIC and MGC.

2. For fatal pathogenic microbes, special care should be taken. One should refer to the technical advices concerning the handling of the microbe(s) under investigation.

3. You should know suitable information about the microbial strain, which you are going to evacuate its cytoplasm (grampositive or negative, spore former, exopolysaccharide producer, yeast, fungi, trophozoit and so on).

4. You should use pure strain. Better to recheck the strain purity.

5. You should know its morphology on plats and under the light microscope.

6. Some microbes are sensitive to the chemical compounds used in the protocol such as P. aeruginosa, which is sensitive to the H2O2 and can be turned to be mucoid (exopolysaccharide producer). For that, it is recommended to use NaOH at the first step.

7. You can change the protocol by using each compound in separate steps (2x strategy). For that, you can simply prepare 2x stock from both of the MIC and the MGC you are going to use.

8. In some special cases where MIC and MGC could not be calculated correctly one can use either the E. coli MIC and MGC or that of another related strains.

9. In the serial dilution step, you should use glass tube to get correct MIC and MGC result. Plastic tubes might give wrong judgment.

10. You should avoid aggregation or clamping, so suitable volume should be used to allow correct contact between the microbial cells and the used chemical compounds.

11. Prepare double (or more) concentration of the MIC and the MGC. Therefore, you can reach the correct concentration after adding the solution which contains the microbe under investigation. In such case it is recommended that one might use the 5x strategy as in the original protocol. However, for the beginner it is recommended to apply NaOH, SDS, H2O2 and CaCO3 using 2x protocol separately.

12. Better to age the microbe to get stronger cell wall and in some cases to reduce the microbe's virulence factors. Alternatively, some chemical compounds can induce cell rigidity.

13. One should monitor the release of the DNA and the protein, spectrophotometrically and by using gel electrophoresis.

14. Use gentle centrifugation not to exceed 3500 rpm.

15. Wash several times after each step to get rid of the residue of the chemical compounds by using saline (0.5-0.9 percentage ) as well as to remove more DNA and protein (etc.).

16. Investigate for the exopolysaccharide producing strain and start the protocol with NaOH to kill them first.

17. You can keep the test tube of the serial dilution for more than one day and you can reevaluate the concentration you use. That because some microbes grow slowly.

18. Calculate the concentration correctly. Any wrong calculation may damage the cells. Light microscope is a suitable tool to monitor the quality of the cells during the adjustment of the MIC and the MGC effect.

19. You can also change the exposing time of the microbe to the used chemical compounds or enzymes. However, regular control for the cell quality by using light microscope should be followed to prevent cell-lysis.

20. You can extend the microbial exposure to H2O2 for overnight.

21. Do not forget that NaOH in the original protocol was used in both experiments (1 and 2) as -1, which mean the MGC. For a certain microbe, you can increase the concentration of the NaOH until the MIC but under the control of the light microscope.

22. After completing the MGs preparation steps, you should examine the cell viability. In case of the existence of viable cells in one or both of the conducted experiments, you should repeat the ghost preparation steps but you still be able to use your incomplete prepared MGs again. There is no need to prepare new cells if the cells are in good quality (investigate the cell quality using the light microscope).

23. In complicated microbes, experimental design should be used. Plackett-Burman, Box Behnken and Excel solver can be used in sequence to get the best-expected optimization. In fact, experimental design could map the critical points involved in the cell ghosts preparation and could be able to optimize perfectly any complicated process. For that MIC for the used compounds in the SL protocol might be used in strains differentiation. Similar strains such as in case of E. coli Bl21 and JM109 prove that they have different MIC and MGC.
Optimizing the gap between MIC and the MGC that can be achieved using Plackett-Burman, Box-Behnken and the Excel solver.

24. Spectrophotometer and electrophoresis can be used to monitor the evacuation of the cytoplasm content.

25. The best MGs are ones which are dead, empty from their cytoplasmic content, have correct 3D structure and are able to induce the immune system upon their treatment.

26. Polyacrylamide gel electrophoresis can be used to show the differences between the viable cells and the ghosts. In original protocol viable cells, which existed after running this protocol (did not turn to dead ghosts and still viable), were subjected to lysis by inducing the lysozyme gene carried on pLysS plasmid.

After the introducing of the critical chemical concentration and activity method for preparing the MGs, many facts were changed and wide range of microbial and mammalian cells as well as other biological containers could be evacuated using such protocol. Therefore, new definition of the MGs can be introduced as follows:

"MGs are empty and dead microbial cells or viruses (envelopes) devoid of cytoplasmic contents or any internal fluidized or any genetic element. That were caused by one or more than one pore happened in their cell walls. Or direct remove of the genetic elements. They have correct 3D structure, morphology and native surface antigens structure able to induce the immune system of the delivered host to produce specific antibodies that could react correctly with the mother viable cell or viruses. Additionally as being empty cells they can be used as drug delivery system for various drugs, genes and antigen or surface antigen expressed protein from another potent pathogenic bacteria. The critical chemical concentration and enzymes activity methods have extended the spectrum of the BGs


Troubleshooting Guide for Cloning

We strongly recommend running the following controls during transformations. These controls may help troubleshoot which step(s) in the cloning workflow has failed.

  1. Transform 100 pg&ndash1ng of uncut vector to check cell viability, calculate transformation efficiency and verify the antibiotic resistance of the plasmid.
  2. Transform the cut vector to determine the amount of background due to undigested plasmid. The number of colonies in this control should be <1% of the number of colonies in the uncut plasmid control transformation (from control #1).
  3. Transform a vector only ligation reaction. The ends of the vector should not be able to re-ligate because either they are incompatible (e.g., digested with two restriction enzymes that do not generate compatible ends) or the 5´ phosphate group has been removed in a dephosphorylation reaction (e.g., blunt ends treated with rSAP). This control transformation should yield the same number of colonies as control #2.
  4. Digest vector DNA with a single restriction enzyme, re-ligate and transform. The ends of the vector DNA should be compatible and easily joined during the ligation reaction, resulting in approximately the same number of colonies as control #1.

The cloning workflow often benefits from an accurate quantitation of the amount of DNAs that are being worked with. We recommend quantification of DNAs whenever possible.


Abstract

Since the discovery of proprotein convertase subtilisin kexin 9 (PCSK9) in 2003, this PC has attracted a lot of attention from the scientific community and pharmaceutical companies. Secreted into the plasma by the liver, the proteinase K–like serine protease PCSK9 binds the low-density lipoprotein (LDL) receptor at the surface of hepatocytes, thereby preventing its recycling and enhancing its degradation in endosomes/lysosomes, resulting in reduced LDL-cholesterol clearance. Surprisingly, in a nonenzymatic fashion, PCSK9 enhances the intracellular degradation of all its target proteins. Rare gain-of-function PCSK9 variants lead to higher levels of LDL-cholesterol and increased risk of cardiovascular disease more common loss-of-function PCSK9 variants are associated with reductions in both LDL-cholesterol and risk of cardiovascular disease. It took 9 years to elaborate powerful new PCSK9-based therapeutic approaches to reduce circulating levels of LDL-cholesterol. Presently, PCSK9 monoclonal antibodies that inhibit its function on the LDL receptor are evaluated in phase III clinical trials. This review will address the biochemical, genetic, and clinical aspects associated with PCSK9’s biology and pathophysiology in cells, rodent and human, with emphasis on the clinical benefits of silencing the expression/activity of PCSK9 as a new modality in the treatment of hypercholesterolemia and associated pathologies.

During 1960 to 1970s, 1,2 it was firmly established that many bioactive secretory proteins, including polypeptide hormones and enzymes, are initially produced as inactive precursors that are transformed into bioactive moieties by limited proteolysis, as illustrated by the processing of pro-opiomelanocortin into adrenocorticotropic hormone and β-endorphin 3 and proinsulin into insulin. 4 This theory led to the concept 5 that conversion of an inactive secretory precursor into active product(s) is catalyzed by a special group of proteases denoted as the proprotein convertases (PCs). These proteases cleave precursors in both the constitutive and the regulated secretory pathways to produce a mature protein/peptide or multiple bioactive fragments. From 1990 to 1999, 8 mammalian PCs were discovered and shown to be responsible for the tissue-specific processing of various secretory precursors. 6,7 Substrates of these PCs include hormones, growth factors, receptors, metalloproteases, membrane-bound transcription factors, and surface glycoproteins. Thus, depending on their site of action and protease activities, 8 PCs are vitally involved in different physiological and clinically relevant processes, 9,10 resulting in activation/inactivation events, some of which affect cardiovascular health. 11

An exhaustive polymerase chain reaction–based screen for a new mammalian PC led to the discovery of the ninth and last member of the family, known as PC subtilisin kexin 9 (PCSK9), which was reported in early 2003. 12 In rodents, PCSK9 was found to be expressed mostly in adult liver hepatocytes, much less so in the small intestine and kidney, and transiently expressed in the developing central nervous system. 12 Because of its rich expression in liver hepatocytes and the localization of its gene (PCSK9) on human chromosome 1p32, a region linked to familial hypercholesterolemia (FH) in some French families, 13 it was suspected and soon confirmed to represent the third FH locus, with the low-density lipoprotein receptor (LDLR) and apolipoprotein B (ApoB) genes being the other 2. 14 Recent reviews have exhaustively documented the history of the discovery of PCSK9 and its relationship to cardiovascular diseases (CVDs). 6,7,15,16

After the discovery of PCSK9 and its relationship to circulating levels of LDL-cholesterol (LDL-C), 12,14 a race was sparked around the world to find not only new gain-of-function (GOF) mutations causing hypercholesterolemia 14,17 but also loss-of-function (LOF) mutations compatible with hypocholesterolemia. 18,19 The highly active Anglo-Saxon D374Y PCSK9 variant is the most remarkable GOF mutation, 20 whereas the heterozygote African nonsense LOF mutation C679X correlates with a remarkable 88% reduction in CVD risk when compared with noncarriers. 21 Such LDL-C–lowering effects are also found in French Canadian subjects with a dominant negative Q152H mutation, which prevents proPCSK9 autocatalytic cleavage into PCSK9 in the endoplasmic reticulum (ER). 12,22 In addition, 2 complete LOF mutations causing marked hypocholesterolemia were found to be compatible with life and result in an amazingly low level of circulating LDL-C levels of ≈0.4 mmol/L. 23,24 Indeed, mammals can survive and stay healthy without PCSK9, as also confirmed in Pcsk9-knockout mice. 25,26 However, this does not seem to be the case in some lower vertebrates, where knockdown of PCSK9 mRNA in zebrafish leads to disorganization of the nervous system and lethality. 27

A third of the adult population in the United States had elevated LDL-C and as a consequence is at risk for CVD. Furthermore, cholesterol-lowering treatment solely based on statins has proven futile in a significant number of patients that are either resistant to statins and do not respond adequately or present serious side effects to these drugs. 28 PCSK9 inhibitors have recently emerged as an alternative new class of cholesterol-lowering drugs. To date, the best studied property of PCSK9 is to bind the hepatocyte-derived LDLR leading to its intracellular degradation. Disrupting this [PCSK9≡LDLR] protein–protein interaction prevents LDLR degradation, thereby raising LDLR levels, lowers LDL-C, 29,30 and is thought to protect from the development of atherosclerosis. 31

Structural and Cellular Biology of PCSK9

PCSK9 Ontogeny, Biosynthesis, Structure, and Degradation of the LDLR

The human 22-kb gene PCSK9 is located on the small arm of chromosome 1p32 and contains 12 exons and 11 introns. 11 The gene encodes a 692–amino acid (aa) proteinase K–like serine protease 6 named PCSK9 14 (originally called neural apoptosis regulated convertase). 6 During rodent development, PCSK9 was shown to be transiently expressed in brain centers, such as the telencephalon, olfactory bulb, and cerebellum. 6 Recent in situ hybridization showed that PCSK9 mRNA is also abundant in the embryonic umbilical artery wall, including presumptive smooth muscle cells and in embryonic membranes (N.G. Seidah, et al, unpublished data, 2014). In the adult, PCSK9 remains highly expressed in liver hepatocytes and less so in the small intestine and kidney. 6

PCSK9 exhibits an atypical zymogen activation pathway when compared with the 8 other members of PC family, 12,32 and its associated activity and biology made it an outlier to classical PCs. The proteinase K–like PCSK9 12 shares sequence similarity with many vertebrate species, including chimpanzee, rhesus monkey, mouse, rat, chicken zebrafish, and ≥40 other species (http://www.ncbi.nlm.nih.gov/). However, although the PCSK9 gene is not found in most invertebrates, it is found in some, such as Branshiostoma floridae, a cephalocordate. 33 Within vertebrates, some species, such as the bovine, do not express the protein. Analysis of the bovine genome revealed the presence of a nonfunctional PCSK9 gene with the absence of 3′ end exons and an early termination at exon 10, 33 suggesting a selection by deletion in some vegetarian species. Practically, this would mean that the fetal bovine serum universally used in cell culture media would not have endogenous PCSK9.

After cleavage of its signal peptide (aa 1–30) in the ER, the zymogen proPCSK9 (aa 31–692) cannot exit this compartment until it intramolecularly cleaves itself at the sequence Val-Phe-Ala-Gln152↓Ser-Ile-Pro (VFAQ152↓SIP) to release the mature enzyme (aa 153–692). Interestingly, proPCSK9 has a tendency to oligomerize in the ER in a disulfide-dependent manner. 12,34 Analysis of the specificity of PCSK9 for autocatalytic cleavage at the P1 Gln152 demonstrated that the only P1 residues that can be recognized by PCSK9 are Gln>Met>Ala>Ser>Thr≈Asn, revealing an unsuspected cleavage specificity. 34 However, different from the other PCs, mature PCSK9 remains noncovalently bonded to its inhibitory prosegment (aa 32–152) and is secreted as a [prosegment≡PCSK9] complex (Figure 1). 12,35 The latter is enzymatically inactive because the prosegment occupies the active site cleft of the protease and shields it from interacting with other substrates (Figure 1). 36 Thus, PCSK9 has no other substrate than itself, and its activity is related to its binding to specific target proteins and to escort the resulting complex toward intracellular degradation compartments. The catalytic subunit of PCSK9 (aa 153–421) contains the active sites Asp186, His226, and Ser386 and the oxyanion hole Asn317, which are typical of all subtilisin-like serine proteases. 6 A small 18-aa hinge region (H aa 422–439) links the catalytic subunit to the C-terminal cys-his–rich domain (CHRD aa 440–692 Figure 1). The first crystal structure of PCSK9 revealed that the CHRD is composed of 3 modules termed M1 (aa 453–531), M2 (aa 530–605), and M3 (aa 604–692), and most of the His residues within the CHRD (9 of a total of 14 His) are found in the M2 domain lining up a groove-like structure. 36,37

Figure 1. Schematic representation of proprotein convertase subtilisin kexin 9 (PCSK9) zymogen processing and binding to the low-density lipoprotein receptor (LDLR). A, The autocatalytic zymogen processing of proPCSK9 (75 kDa) at Val-Phe-Ala-Gln152↓Ser-Ile-Pro (VFAQ152↓SIP) into the [prosegment (15 kDa)≡PCSK9 (62 kDa)] complex is emphasized, together with the positions of the active site Asp186, His226, and Ser386 and the oxyanion hole Asn317. The C-terminal hinge domain (H) and cys-his–rich domain (CHRD) are shown. B, Cartoon representation of the cell surface interaction of the catalytic domain of PCSK9 with the epidermal growth factor-A (domain of the LDLR, as well as the suspected interaction of the prosegment with the β-barrel domain of the LDLR and the CHRD with a putative membrane-bound protein X. C, Crystal structure of the ectodomain of the LDLR with PCSK9 emphasizing the interaction between them and the 3 subdomains in the CHRD (M1, M2, and M3). The interaction of protein X is presumed to be with one of the latter subdomains, possibly M2.

The best characterized activity of the [prosegment≡PCSK9] complex is its ability to bind to specific target proteins and to escort them toward intracellular degradation compartments. The first PCSK9 target to be identified is the LDLR at the surface of liver hepatocytes. 35,38,39 The catalytic subunit of PCSK9 was shown to bind the epidermal growth factor-A (EGF-A) domain of the LDLR (Figure 2), 40,41 as well as the similar domain found in other LDLR superfamily members (eg, very LDLR [VLDLR], apolipoprotein E receptor 2 [ApoER2], 42,43 and lipoprotein receptor–related protein 1 [LRP1] 44 ). Normally, the [LDLR≡ LDL-C] complex enters cells via clathrin heavy chain–coated vesicles, and when internalized, the acidic pH of endosomes causes the allosteric dissociation of the LDLR and its recycling to the cell surface, whereas the LDL-C is directed to lysosomes for degradation, where cholesterol is recovered and distributed in the cell (Figure 2). 45 In contrast, the complex [PCSK9≡LDLR], although also entering the cells via clathrin-coated vesicles, 46,47 does not dissociate at acidic pHs but is rather more tightly associated, 36 and, through some unknown mechanism it is escorted to lysosomes for degradation by as yet undefined proteases. 35,46 An added complication is the observation that PCSK9 can enhance the degradation of the LDLR either by a direct intracellular pathway not requiring its secretion 48 or extracellularly upon binding the LDLR at the cell surface (Figure 2). 49 Clathrin light chains are not required for clathrin-mediated endocytosis but are critical for clathrin-mediated trafficking between the trans Golgi network and the endosomal system. 50 Indeed, clathrin light chain siRNAs that block direct intracellular trafficking from the trans Golgi network to lysosomes rapidly increased LDLR levels within the human hepatocellular carcinoma cell line HepG2 cells in a PCSK9-dependent fashion, without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. 48 Whether these 2 pathways are operative in all tissues is still not clear. In support of this model, a recent observation revealed that PCSK9 lacking the M2 domain of the CHRD can still degrade the LDLR intracellularly but not when added outside cells, supporting the presence of 2 distinct sorting and regulatory pathways of the [PCSK9≡LDLR] complex. 51

Figure 2. Schematic representation of the intracellular and extracellular pathways of proprotein convertase subtilisin kexin 9 (PCSK9) induced degradation of the low-density lipoprotein receptor (LDLR). When PCSK9 levels are high or if it has a gain-of-function, it will enhance the degradation of the LDLR using both the intracellular and the extracellular pathways leading to the degradation of the (PCSK9≡LDLR) complex in lysosomes. This results in low levels of the LDLR at the cell surface and increased levels of circulating LDL-C. In absence or under low levels of PCSK9, cell surface LDLR levels are high and the LDLR can be recycled back to the surface after delivery of LDL particles to acidic endosomes. The evidence for the intracellular pathway is based on the knockdown of clathrin light chains in the human hepatocellular carcinoma HepG2 cells. 48,50 The extracellular pathway-specific treatments include the use of a monoclonal antibody (mAb), an inhibiting adnectin or a small-molecule epidermal growth factor-A (EGF-A)–like inhibitor. TGN indicates Trans Golgi Network.

Interestingly, both the intracellular and the extracellular LDLR degradation activities of PCSK9 require the presence of the CHRD because the [PCSK9-ΔCHRD≡LDLR] complex that lacks this domain, although still capable of internalization into endosomes, does not traffic to lysosomes and is likely recycled to the cell surface. 51,52 This has led to the search of other proteins that may interact with the CHRD and the LDLR and drive the [PCSK9≡LDLR] complex to lysosomes.

Recent reports shed some light on this still obscure mechanism. Two studies suggest that the CHRD binds weakly ≥1 of the 7 Ca 2+ -coordinating repeats in the N-terminal ligand-binding domain of the LDLR 53 and that such binding may be necessary to prevent the dissociation of the [PCSK9≡LDLR] complex in acidic endosomes. 54 Furthermore, it was suggested that the binding of the positively charged CHRD to the negatively charged LDLR’s ligand-binding domain is favored at acidic pHs, 53,54 where the His residues of the CHRD would be positively charged. These data support the finding that aside from the EGF-A domain, ≥3 of the 7 ligand-binding repeats of the LDLR and the β-propeller domain are necessary for the LDLR to be degraded in the presence of PCSK9. 52 However, such a binding is likely to be weak because all reported crystal structures of PCSK9 at either neutral or acidic pH do not reveal any interaction of the CHRD with the N-terminal repeats (aa 25–313) of the LDLR. One exception is the predicted weak hydrophobic interaction of Leu626 of the β-propeller domain of the LDLR with Leu108 of the prosegment of PCSK9, 41 which was also deduced from a GOF L108R PCSK9 mutant that possibly strengthens this interaction by favoring the electrostatic binding of Glu605 of the LDLR to the mutant Arg108 of PCSK9. 17

Nevertheless, because the cytosolic tail of the LDLR is not necessary for the sorting of the [PCSK9≡LDLR] complex to lysosomes, 44,55 this suggests that another protein must bind the luminal CHRD domain and that such protein X would also have a transmembrane domain and cytosolic tail linking motor proteins in the cytosol to direct the complex to lysosomes (Figure 1). 44 In that context, a recent report proposed that the amyloid precursor–like protein-2 (APLP-2) can bind the CHRD at the surface of cells and in endosomes, and that this [LDLR≡PCSK9≡APLP-2] tripartite complex is then targeted to lysosomes (Figure 2), 56 by a still undefined mechanism. 57 Does APLP-2 and other members of the family, such as amyloid precursor protein and APLP-1, represent the missing link(s) to understand the cellular trafficking of the [PCSK9≡LDLR] complex to lysosomes? It is still too early to settle this point because in vivo analyses of Aplp2-knockout mice are still lacking, and our preliminary data suggest that knockdown of APLP-2 expression in cells does not affect the ability of PCSK9 to degrade the LDLR or LRP1 44 both intracellularly and extracellularly (M. Canuel, et al, unpublished data, 2014).

What is the role of the prosegment in the sorting or activity of the [prosegment≡PCSK9] complex? First, the human Q152H-dominant negative variant confirmed that the autocatalytic cleavage of proPCSK9 into PCSK9 is an absolute requirement for exit of this protein from the ER 12 and its subsequent enhancement of the LDLR degradation. 22,34 Second, because the [Δ31-58-prosegment≡PCSK9] complex lacking the N-terminal acidic sequence of the prosegment (aa 31–58) is ≥4- to 7-fold more active in degrading the LDLR, 58,59 this suggests that this acidic region, which is likely not stabilized on its own because it is not seen in any crystal structure reported, is a negative regulator of the activity of PCSK9 on the LDLR. Recent evidence suggested that the association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLR, thereby blunting PCSK9-mediated LDLR degradation. 60 Because ApoB is the major protein in LDL, this suggests that it is the active component that binds PCSK9 in plasma and blunts its function on the LDLR. Whether such binding implicates the interaction of the acidic aa 31 to 58 at the N terminus of the prosegment of PCSK9 with a positively charged domain in ApoB has yet to be demonstrated. Cocrystallization of PCSK9 with the ApoB-binding domain should shed light on this model.

The secretory pathway of eukaryotic cells packages cargo proteins into Coat Protein-II–coated vesicles for transport from the ER to the Golgi, which are then sorted into other organelles or secreted. What are the other cellular partners of PCSK9 that regulate its exit from the ER and trafficking? It was recently reported that the exit of the complex [prosegment≡PCSK9] from the ER requires an interaction with a putative membrane-bound protein that links via its tail the cytosolic protein sec24a that is associated with COP-II vesicles. 61 The absence of the latter results in markedly lower levels of secreted PCSK9, leading to higher levels of hepatic LDLR protein levels because of decreased degradation. It would be stimulating to find out if the opposite also exists (ie, that some mutations in PCSK9 or its putative ER partner may enhance PCSK9 secretion from the ER).

In conclusion, the escort and degradation of the [PCSK9≡LDLR] complex is regulated by a variety of proteins, including PCSK9 and LDLR themselves, protein X, ApoB, sec24a, and most likely, other undefined and transitory partners that would interact with this complex along the secretory route, even as early as the ER. 46,62 Furthermore, the PC Furin cleaves PCSK9 at Arg218↓ at the surface of hepatocytes and likely results in its in vivo inactivation, 63,64 suggesting that some proteases could regulate PCSK9 activity. PCSK9 seems to be unique when compared with other PCs in the sense that it is the only convertase that has only 1 enzymatic substrate, itself. It seems that this relatively more recent and polymorphic convertase was selected for its protein–protein interaction with LDLR-like receptors, rather than as a protease, because the inhibitory prosegment remains tightly bound to the catalytic subunit. Whether factors exist, other than the prosegment, that could also inhibit the enzymatic activity of PCSK9 is not yet known. The evolutionary conservation of this enzyme suggests that such a regulatory mechanism has been maintained in most vertebrates but not in invertebrates that express other PC-like proteases.

Other PCSK9 Target Proteins

Although the LDLR is no doubt the best studied target of PCSK9 and probably relevant physiologically because it controls the levels of circulating LDL-C PCSK9 was also found to escort other receptor members of the LDLR superfamily toward endosomal/lysosomal degradation. Thus, PCSK9 was first found to enhance the extracellular and intracellular degradation of the closest LDLR family members, namely the VLDLR and ApoER2 (LRP8). 42,43 However, although the interaction of the catalytic domain of PCSK9 with the EGF-A–like domains of these receptors was confirmed, the specific aa in each protein implicated in such interactions are not the same as for the LDLR. For example, in contrast to the LDLR, the GOF D374Y PCSK9 does not degrade these other receptors more efficiently than wild-type PCSK9. 42 The receptors LDLR, VLDLR, and ApoER2 have been confirmed as PCSK9 target proteins in mice 25,26,65,66 and the LDLR in monkeys 67 and human. 68 Recently, we showed that PCSK9 can enhance the degradation of LRP1 in various cells 44 although proof of this activity in vivo is still lacking. Finally, CD36, a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFA), was also suspected to be a PCSK9 target in intestinal epithelial cells 69 and adipose tissue. 65 Recently, this PCSK9 activity on CD36 was elegantly proven in cells and adipocytes. 70 because the structures of CD36 or CD81 (see below) do not exhibit an EGF-A–like domain, their respective sequences that bind PCSK9 either directly or indirectly have yet to be defined.

The fact that many PCs can process surface glycoproteins of infectious viruses 6 prompted us to test the effect of the lack of PCSK9 on the titer and infectivity of viruses that infect the liver (richest source of PCSK9), 12 such as the hepatitis C virus (HCV). The data showed that PCSK9 targets 2 hepatic HCV receptors for degradation, namely the LDLR and the tetraspanin protein CD81. 71 Furthermore, it was recently reported that other viruses bind to cell surface LDLR family members to enter and infect cells, including vesicular stomatitis virus that likely uses the LDLR and LRP1 as entry receptors. 72 These results suggested that although inhibiting PCSK9 may be beneficial to reduce the levels of circulating LDL-C, it potentially could enhance the infectivity of certain viruses, such as HCV, vesicular stomatitis virus, the common cold rhino virus, and rous sarcoma virus.

It was suggested that PCSK9 could enhance the degradation of certain targets within the ER/ER-Golgi intermediate compartment. Two examples were reported: (1) The Alzheimer disease-associated aspartyl protease β-secretase β-amyloid precursor protein cleaving enzyme-1 (BACE1) is transiently acetylated on 7 Lys residues in the lumen of the ER/ER-Golgi intermediate compartment. The acetylated intermediates of the nascent protein are able to reach the Golgi apparatus, whereas the nonacetylated ones are retained and degraded in a post-ER compartment. PCSK9 was reported to contribute to the disposal of nonacetylated BACE1. 73 This interesting observation still requires in vivo validation. (2) In the second example, within the ER/ER-Golgi intermediate compartment of cells, PCSK9 was reported to enhance the degradation of the epithelial Na + channel (ENaC) that is critical for Na + homeostasis and blood pressure control. 74 This observation was intriguing, especially in view of the expression of PCSK9 in the kidney. 12 However, our data revealed that in PCSK9-knockout mice, the basal and angiotensin-II–induced blood pressure rise is not different from wild-type controls, suggesting that EnaC levels are not appreciably increased in the absence of PCSK9, at least in mice (N.G. Seidah and T. Reudelhuber, unpublished data, 2014). These results cast some doubt as to the physiological relevance of the reported cellular role of PCSK9 on epithelial Na + channel. 74

Gene Regulation of PCSK9

Transcriptional Regulation of PCSK9 Expression

As for any other gene, regulation of PCSK9 gene expression begins at transcription. A stringent scanning of the proximal promoter of the PCSK9 gene (600 bp upstream region and first exon) was conducted to search for probable transcriptional regulatory elements using the Nsite online algorithm (http://linux1.softberry.com). The functional characterizations of these elements have focused mostly on the human and mouse PCSK9 gene promoter.

Nuclear-Binding Factors

The sterol regulatory element (SRE) is the most conserved of these transcriptional motifs, consistent with a modulatory role of this gene in cholesterol metabolism. SRE is the binding site for SRE-binding proteins (SREBPs), the master transcriptional factors in lipid biosynthetic pathways. Shortly after the discovery of PCSK9, an unbiased analysis of the hepatic transcriptome of mice showed that the level of PCSK9 mRNA was strongly downregulated when mice were fed a cholesterol-rich diet and upregulated in transgenic mice overexpressing nuclear SREBP-1a or SREBP-2. 75 Later, Dubuc et al 76 showed that statins, which inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, resulting in a feedback activation of nuclear SREBP-2, increased the level of PCSK9 mRNA in HepG2 human hepatocytes in culture. The statin induction could be abrogated by mevalonate, a post–3-hydroxy-3-methyl-glutaryl-CoA reductase cholesterol precursor, confirming the link of this regulation to cholesterol metabolism. 76 SREBP-1c was also implicated in postprandial insulin upregulation of PCSK9 gene expression in hepatocytes because constitutive expression or inactivation of this transcriptional factor increased or reduced the level PCSK9 mRNA in these cells in culture, respectively. 77 SREBP-1 and SREBP-2 have been shown to bind to the PCSK9 gene promoter SRE in vitro specifically. 78

SREBP activation of the PCSK9 gene promoter is potentiated by the hepatocyte nuclear factor-1α (HNF1α), which binds to an element located 28 nucleotides (nts) upstream of the SRE. This site is conserved between primate and rodent PCSK9 promoters and is absent in the LDLR promoter. Its invalidation by site-directed mutagenesis dramatically reduces PCSK9 gene promoter-driven expression of a reporter gene in transfected HepG2 cells. 79 Upregulation of HNF1α expression by statins 80 contributes to sustained PCSK9 production/secretion that attenuates the LDLR-mediated clearance of plasma LDL-C induced by these drugs. Its expression is downregulated by the phytochemical berberine 79 and by activators of hepatic mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, 81 making such compounds potential enhancers of the LDL-C clearance.

Situated between the SRE and HNF1α-binding motif is a histone 1 nuclear factor P(H1NFP)–binding motif that has been shown to be critical to the functionality of the SRE in the PCSK9 promoter, being required for both basal and enhanced activation of this promoter by SREBP-2. Binding to this motif, H1NFP cooperates with the cofactors nuclear protein of the ataxia telangectasia mutated locus and transformation/transactivation domain–associated protein to activate the PCSK9 promoter. Transformation/transactivation domain–associated protein is a cofactor of histone acetyltransferase, which mediates histone H4 acetylation on the promoter, facilitating its activation. 82 Inversely, impaired acetylation or deacetylation of histone at the PCSK9 promoter can hamper its activation. Sirtuin 6, an NAD + -dependent histone deacetylase is a transcriptional repressor of the PCSK9 because its absence in mouse liver leads to increased expression of this gene, whereas its overexpression reduces it. 83 The insulin-responsive element–binding factor known as FoxO3 recruits Sirtuin 6 to the PCSK9 promoter, where it deacetylates histone 3 and causes local chromatin changes that negate promoter activation. Interestingly, the recognition element of FoxO3 is embedded within that of HNF1α. Interactions between the 2 oppositely acting factors have been demonstrated in coimmunoprecipitation studies. 83 The outcome of this competitive interaction may depend on the relative amounts of these factors and their required cofactors.

Nuclear Receptors

The PCSK9 promoter is also regulated by ligand-activated nuclear receptors, such as Farnesoid X receptor and peroxisome proliferator–activated receptors (PPARs). Farnesoid X receptor, which binds chenodeoxycholic acid, has been implicated in the fall of PCSK9 mRNA in cultures of hepatocytes after exposure to this bile acid component. This implication is probably indirect because siRNA-induced knockdown of the receptor was ineffective. 84

PPARα agonists, such as fenofibrate, are commonly used to treat hypercholesterolemia and mainly hypertriglyceridemia. Their effects on PCSK9 mRNA levels have been explored with mixed results. They have been shown to repress PCSK9 gene expression in a human hepatocyte cell line and isolated mouse hepatocytes, 85,86 but the WY14643 agonist had no effect on the level of PCSK9 mRNA in isolated hamster hepatocytes. 87 The reasons for this discrepancy are unclear as are those from clinical studies in fibrate-treated patients, showing increased plasma PCSK9 levels in most studies 88–91 and a decreased levels in another. 85

PPARγ ligands, 15d-PGJ1 or pioglitazone, have been shown to augment the level of PCSK9 mRNA in HepG2 cells. This induction can also be achieved by preventing phosphorylation of the receptor by extracellular-regulated kinases 1 and 2 with such specific inhibitors PD98059 or U0126. 92

It should be noted that, in this transcriptional regulatory network, the SREBP-2 and HNF1α genes themselves are often downstream targets of other factors. For example, PPAR (α or γ) activation in liver, hepatocytes, or enterocytes has been shown to reduce the level of nuclear SREBP-2 and ultimately of cholesterol biosynthesis. 93,94 mTOR1 also downregulates HNF1α, resulting in reduced PCSK9 transcription. 81 Sirtuin 6 had similar inhibitory effects on SREBP-2 and its target genes. 95,96 Thus, SREBP-2 and HNF1α seem to stand at a crossroad of transcriptional pathways regulating expression of genes that could influence intracellular cholesterol homeostasis, including PCSK9 and LDLR genes.

Thus, transcriptional up- or downregulation of PCSK9 is determined by the relative abundance and activity of a variety of nuclear factors acting in cooperation or in competition on cis regulatory elements. The elements described above are located in the proximal promoter, but the possibility of more distal ones cannot be discounted as yet. The functionality has been explored primarily in liver cells. A better understanding of transcriptional regulation may allow one to anticipate the direction of PCSK9 expression in these cells under certain physiological or therapeutic conditions. Furthermore, it may clarify why some mutations in the cognate gene are associated with altered expression of the protein, as has been observed with c.-332C>A variant located near the SRE and associated with a GOF phenotype in a Spanish population. 97

Post-Transcriptional Regulation of PCSK9 Expression

Regulation of mRNA stability and translation into protein are more immediate response mechanisms to physiological demands than transcription. To our knowledge, there has been no experimental evidence of PCSK9 expression regulation at these levels. However, the sequences of the 3′ untranslated region (3′-UTR) of its mRNA exhibit features suggestive of such a regulation. Structurally unstable mRNAs are typically characterized by a high AU content in their 3′-UTR. 98 At the extreme end of 1.3-kb long 3′-UTR of human PCSK9, mRNA is an island of ≈120 nts (nts 575–3692 NM_174936.3), which exhibits 71% AU content, contains 2 AUUUA canonical AU-rich elements commonly associated with mRNA instability, and is relatively conserved among primates and rodents. The relevance of this AU island in mRNA stability remains to be functionally determined. This is further justified by the fact that the mRNA of the LDLR, the opposing partner of PCSK9 in lipid homeostasis, is itself, subject to downregulation through its 3′-UTR by heterogeneous nuclear ribonucleoprotein D. 99,100 In addition, the presence of a potential miRNA-24 recognition in the 3′-UTR of human PCSK9 mRNA warrants investigation of its possible regulatory role, considering the growing implications of miRNAs in cholesterol metabolism. 101

PCSK9 Animal Models

Hypercholesterolemia and Atherosclerosis (Transgenic Models)

Adenoviral-mediated expression of human PCSK9 in mice results in an intermediate LDLR-knockout phenotype. 38 PCSK9 effect is mostly exerted in a paracrine/endocrine fashion as shown by parabiosis experiments. 102 As expected, transgenic mice overexpressing PCSK9 under an Apoe promoter [Tg(Apoe-PCSK9)] are viable, fertile, and severely hypercholesterolemic. 26,39 Interestingly, although the LDLR is not detected in the liver of these mice, the circulating LDL-C is only increased ≈5-fold when compared with ≈15-fold in Ldlr-knockout mice. 26 This suggests that the LDLR in tissues other than liver contribute to the clearance of circulating LDL-C, and that the LDLR in these extrahepatic tissues are not regulated by, or accessible to, circulating PCSK9. Possible mechanisms are described in the section below describing the role of PCSK9 in extrahepatic tissues.

On an Apoe-knockout genetic background, Tg(Pcsk9) mice develop accelerated atherosclerosis with larger plaque size, when fed a regular chow diet, and a protective effect was seen with Pcsk9-knockout mice on the same background. 31 The late complications of atherosclerosis in the form of vascular calcified plaques were also observed in these Apoe-knockout/Tg(Pcsk9) mice but to a lesser degree and with a longer latency when compared with Ldlr-knockout mice. 103 Thus, these ApoE-deficient Tg(PCSK9) mice exhibit an intermediate phenotype to the complete absence of the LDLR and are, therefore, more relevant to the majority of individuals with an attenuated LDLR activity and dyslipidemia. Finally, hypercholesterolemia and atherosclerosis were recapitulated in a pig model of a human PCSK9 GOF D374Y mutant. A liver-specific expression of this mutant in minipigs resulted in markedly reduced levels of hepatic LDLR, impaired LDL-C clearance, severe hypercholesterolemia, and eventually these minipigs developed spontaneous progressive atherosclerotic lesions that could be visualized by noninvasive imaging. 104 Two conclusions can be drawn from these observations: first, inhibition of PCSK9 would be a potential therapeutic modality second, the use of large species models will better advance the translational research aspect of treating atherosclerosis from animals to human. 104

Hypocholesterolemia and Fat Metabolism (Knockout Models)

One of the classical experiments aimed at understanding the functions of a given gene is to delete it from the genome and observe the consequences of such lack of expression. Complete Pcsk9-knockout resulted in viable and fertile mice exhibiting severe hypocholesterolemia, 25,26 with an ≈40% and ≈80% drop in total cholesterol and LDL-C, respectively. 18 In comparison, liver-specific Pcsk9-knockout mice exhibit ≈27% less circulating cholesterol, suggesting that liver PCSK9 contributes to ≈70% of the cholesterol phenotype. 18 Interestingly, the liver-specific loss of PCSK9 expression 26 resulted in its complete absence from circulation, demonstrating that hepatocytes are the primary source of plasma PCSK9. 65 Notably, lipidomics analyses of the plasma of Pcsk9-knockout and transgenic mice revealed that Pcsk9-knockout mice result in a marked reduction in the plasma levels of sphingomyelin and ceramides, which are known risk factors for coronary artery disease. 105 These lipid biomarkers should now also be measured in the plasma of patients treated with inhibitors of cholesterol biosynthesis (eg, statins) or anti-PCSK9 drugs to evaluate the efficacy of treatment because it was also shown that similar reductions are seen in the plasma of individuals with the R46L LOF mutation 105 or those treated with simvastatin. 106

As mentioned above PCSK9 may target other receptors, and VLDLR is upregulated in perigonadal depots of female Pcsk9-knockout mice. As a consequence, adipocytes became hypertrophic because of higher levels of adipocyte VLDLR and increased uptake of FFA, 65 possibly via an associated increased level of CD36. 70 Conversely, plasma triglycerides were slightly increased in Pcsk9-knockout males (+35% not significant) and females (+46% P=0.03). Furthermore, in vivo studies revealed that PCSK9 deficiency was associated with a 2-fold decrease in postprandial triglycerides levels, suggesting improved triglycerides clearance and a role for PCSK9 in triglycerides metabolism, 107 possibly via its ability to enhance the degradation of VLDLR and CD36. PCSK9 is thus essential in fat metabolism because it maintains high circulating LDL-C levels via hepatic LDLR degradation, but at the same time it downregulates triglycerides and FFA entry into visceral adipocytes, possibly via adipose tissue VLDLR 65 and CD36 degradation. 69

Role of PCSK9 in Extrahepatic Tissues

Adipocytes, Muscles, and Myocytes

PCSK9 is mainly expressed in adult liver, small intestine, kidneys, 12,26 and pancreas. 12,108 Although not expressed in adipocytes, circulating PCSK9 produced by the liver can regulate the levels of cell surface receptors in this tissue. 65 Because human PCSK9 targets ex vivo human VLDLR 42 and binds in vitro mouse VLDLR, 43 PCSK9 has the capacity to target human VLDLR in vivo. Indeed, visceral fat accumulates in PCSK9-deficient mice because of the high uptake of chylomicrons and VLDL-C in tissues that predominately express the VLDLR (such as adipose tissue followed by heart and muscles). Lack of PCSK9 increases the surface expression of the VLDLR that facilitates triglycerides hydrolysis and FFA uptake in the visceral adipocytes. In theory, muscle and heart tissues would have been affected too, but muscles burn fat continuously, whereas adipose tissues tend to store energy in the form of fat droplets for later usage. Lack of PCSK9 in mice or LOF PCSK9 variants in humans are suggested to affect postprandial lipemia, 107 which further establishes the association between serum PCSK9 and lipid metabolism. At least in Pcsk9-knockout mice, no upregulation of cytokines, associated with the metabolic syndrome, was observed. 65 Whether an increased visceral fat deposition also occurs in humans lacking functional PCSK9 remains to be elucidated. Perigonadal fat is part of what is called the visceral adipose tissue, which correlates directly with obesity-related metabolic disease and coronary heart disease. However, it was recently reported that in patients with obesity with similar levels of visceral adipose tissue, metabolic complications were more prevalent in those exhibiting higher intrahepatic triglycerides. 109 In a clinical perspective, because Pcsk9-knockout mice do not develop liver steatosis 26 and are not prone to obesity, administration of a PCSK9 inhibitor for the treatment of hypercholesterolemia is not expected to result in adverse effects.

Adrenals and Kidneys

Like liver, adrenals require synthesis and uptake of cholesterol for proper function. It was found that annexin A2 is a natural extrahepatic inhibitor of PCSK9 110 and that in Anxa2-knockout mice, plasma PCSK9 doubles and LDLR decreases by ≈50% in some extrahepatic tissues, such as adrenals, small intestine, and colon but not in liver. 111 In addition, transgenic mice expressing human PCSK9 mainly in the kidney resulted in LDLR degradation mostly in the liver but not in adrenals. 112,113 Thus, it is probable that the high levels of annexin A2 in adrenals are responsible for the refractoriness of the LDLR in this tissue to the action of PCSK9. 110,111 Another, not mutually exclusive, possibility is that in refractory tissues, such as the adrenals, the complex [PCSK9≡LDLR] enters endosomes but does not traffic to lysosomes and may actually recycle back to the cell surface. Accordingly, a possible explanation for the tissue-specific activity of PCSK9 could be that extrahepatic tissues that do not respond to PCSK9 are unable to sort the [PCSK9≡LDLR] complex to lysosomes, likely because of unfavorable cellular response or absence of functional specific protein(s) that control lysosomal targeting of the complex (Figure 2). In support of this notion, it was recently shown that fibroblasts, which are resistant to PCSK9-induced degradation of the LDLR, can bind PCSK9 but that the endocytosed PCSK9 dissociates from the LDLR within early endosomes, and the latter is rapidly recycled back to the cell surface and not sent to lysosomes for degradation. 114 Alternatively, some tissues, such as adrenals, are enriched in an endogenous inhibitor of PCSK9 (eg, annexin A2), that prevent its function on the LDLR. 111

The physiological role of PCSK9 in kidney is still not clear, especially because its expression therein shifts from the cortex in the embryo to the lumen in adult rodents. 12 It is also possible that PCSK9 acts locally in some extrahepatic tissues that express this protein (eg, small intestine, pancreas, and kidney), and that its circulating levels would not affect the LDLR in these tissues. The reported possible PCSK9-induced degradation of epithelial Na + channel in kidney cells 74 needs in vivo confirmation because basal blood pressure and angiotensin-II induced rise in blood pressure in mice are not modified by the lack of expression of PCSK9 (N.G. Seidah and T. Reudelhuber, unpublished data, 2014).

Small Intestine and Pancreas

Findings from cellular and tissue expressions from rat, mouse, and human showed that not only the liver and cerebellum are rich in PCSK9 but also the small intestine, especially the ileum. 12 PCSK9 regulation of the LDLR protein levels in intestinal cell lines has been reported. 69,115 The possible implication of PCSK9 in the transintestinal reverse cholesterol excretion from the small intestine has been recently evoked. 116 Although the LDLR is involved in this process, it seems that yet another receptor(s) sensitive to PCSK9 is/are also implicated. 116 Could this receptor be one of the known targets of PCSK9, such as VLDLR, apoER2, LRP1, CD36, or yet another receptor to be identified in the future? Finally, it was reported that the N-terminal negatively charged sequence of the prosegment of PCSK9 is critical for the binding of the [prosegment≡PCSK9] to LDL-C (likely to a positively charged sequence within ApoB). 60 Because liver expresses mostly ApoB-100 and the small intestine ApoB-48, and the latter does not bind the LDLR, it is possible that the difference between liver and small intestine in relation to responsiveness to circulating PCSK9 may be affected by these 2 isoforms of ApoB.

PCSK9 was originally observed to be highly expressed in pancreatic β-cell lines. 12 When compared with controls, older male Pcsk9-knockout mice express more LDLR in pancreatic islet cells and are glucose-intolerant. 108 It remains to be seen whether circulating PCSK9 affects the LDLR and other receptors at the surface of β-cells, and whether the upregulation of these in the absence of endogenous PCSK9 or from circulation results in lipotoxicity or impairment of β-cell function.

PCSK9 and Pathological Conditions

Liver Regeneration

Given that PCSK9 is mainly produced by the liver and that the latter is a regenerating organ, it was expected that conditions that perturb liver function may influence PCSK9 levels and that the latter may in turn affect the ability of the liver to recover from certain insults. In vivo studies revealed that after partial hepatectomy, regenerating livers of Pcsk9-knockout mice exhibited the presence necrotic lesions and a significant delay in their regenerating capacity. 26 Fortunately, this lesion phenotype and regenerating capacity were reversed when a high-cholesterol diet was supplemented to mice before and after partial hepatectomy. 26 These results were the first indication that on hepatic damage, patients lacking PCSK9 could be at risk. However, under normal circumstances, lipid accumulation in hepatocytes of these mice was markedly reduced under both regular and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. 26 In conclusion, although PCSK9 inhibition may protect against the development of liver steatosis, it may also result in an increased risk of liver damage after a liver insult. It remains to be seen what kind of insults are more deleterious to liver function in the absence of PCSK9 (eg, hepatitis, liver cirrhosis, or hepatocellular carcinoma).

Insulin Resistance

Adult PCSK9-deficient male mice exhibit impaired glucose tolerance and may be at risk to develop diabetes mellitus on aging. 108 However, the underlying mechanism is largely unknown. In 1 population study, it was shown that subjects with PCSK9 LOF R46L have a significant increment in markers of insulin resistance, namely insulin, homeostasis model assessment of insulin resistance, and the adipokine hormone leptin in individuals carrying 1 copy of apoE2 when compared with those carrying other apoE isoforms. 117 This indicates that heterozygote LOF of APOE and PCSK9 may lead to insulin resistance. To date, the only woman analyzed that lacks completely PCSK9 because of compound heterozygous null mutations 23 has not been reported to have insulin resistance.

Viral Infections

HCV particles interact with a number of putative HCV receptors, including CD81, scavenger receptor class B type I, and Claudin-1. 118 Circulating HCV particles are associated with VLDL and LDL of infected patients, suggesting that the LDLR is critical for hepatic viral infection. Recently, it was shown that liver expression of CD81 is markedly increased in Pcsk9-knockout mice and that the HCV receptor CD81 protein is downregulated independently from the LDLR. 71 Therefore, it was proposed that the plasma level and activity of PCSK9 could modulate HCV infectivity in humans. In summary, the LDLR and CD81, 2 HCV entry receptors are dose dependently downregulated by PCSK9, resulting in the reduction of the cellular infectivity of HCV in mice. Although never shown in humans yet, PCSK9 has the potential to protect against HCV. Therefore, caution must be exercised when administering PCSK9 inhibitors to subjects that could be potentially infected with HCV or other viruses that use ≥1 of the LDLR superfamily members as entry receptors. 72

Physiological Modulation of PCSK9 Levels

Variation with Age, Sex, and Pregnancy

A cross-sectional pediatric study of ≈1700 subjects revealed that in boys, plasma PCSK9 levels continuously decreased from age 9 to 16 years, correlating with mean total cholesterol levels that continuously decreased with age. In contrast, in girls, PCSK9 levels peaked at the age of 13 years and then decreased to higher levels than boys at 16 years, and total cholesterol levels were higher in 9 and 16 years olds than in 13 years olds. 119 It is thought that this regulation follows a growth hormone pattern. 119 In the multiethnic Dallas Heart Study of 3138 patients, plasma PCSK9 levels were significantly higher in women (n=1863) than in men (n=1489). This difference persisted after adjusting for most variables.. 120 In the same study, premenopausal women had considerably higher levels of plasma PCSK9 than postmenopausal women. Estrogen treatment did not significantly affect fasting PCSK9 levels in postmenopausal women. In comparison, there was no difference in plasma PCSK9 levels in men >50 versus those <50 years of age. Similar findings were obtained in older men and postmenopausal women, with 9% to 14% higher PCSK9 levels observed in women. Finally, serum PCSK9 levels were increased in pregnancy at term. However, recent data also showed that the human fetus has ≈2-fold lower PCSK9 levels than the mother, which may be necessary to provide LDL-C to the growing fetus. 121

Variation With Diet and Medication

It was found that a 5-week Mediterranean diet in normal subjects can lower plasma LDL-C and PCSK9 by as much as ≈10% and ≈15%, respectively. 122 Moreover, it was shown that PCSK9 transcription could be suppressed by fasting and induced by insulin, likely by activating liver X receptor and SREBP-1c. 77 Like the LDLR, PCSK9 is also upregulated by intracellular sterol depletion and statin treatment. Thus, in hepatic cell lines, statins coordinately upregulated the mRNA expression of LDLR and PCSK9. 76 This suggested that statins would have a higher capacity to decrease LDL-C if not for the associated rise in PCSK9 level after statin. Indeed, statin treatment of Pcsk9-knockout mice results in a higher reduction in LDL-C than that of wild-type mice, signifying a hypersensitivity state. 25 This was confirmed in individuals with FH harboring the common LOF R46L variant who seem to be more responsive to statin. 123 Although statins directly increase PCSK9 mRNA expression, PPARα agonists, such as fibrates, indirectly affect PCSK9 expression through modulation of cholesterol levels. 88 In a randomized trial, although statins were shown to increase the levels of circulating PCSK9, the cholesterol absorption inhibitor ezetimibe had no effect on PCSK9. 124 Finally, in a study in which women underwent in vitro fertilization, high estrogens resulted in a reduction in VLDL, LDL, and PCSK9 levels. 125 However, this might have been an effect of the 3-fold increase in growth hormone in response to induction of ovulation. Thus, growth hormone may negatively affect PCSK9 to favor increased uptake of circulating cholesterol by growing cells.

PCSK9 Genetic Variance

Family studies of patients with coronary heart disease led to the mapping of PCSK9 gene to FH, with GOF mutations exhibiting high serum levels of LDL-C 14 likewise LOF mutations were later found to lower LDL-C and could protect from coronary heart disease. 18 Until now, only 2 women were reported to lack PCSK9 completely and to have low levels of circulating LDL-C: a subject with compound heterozygote LOF mutations 23 and 1 homozygote LOF C679X mutation. 24 A continuously updated list of all natural mutations of PCSK9 can be found in (http://www.ucl.ac.uk/ldlr/LOVDv.1.1.0/index.php?select_db=PCSK9), and some of them were recently summarized. 126

Natural Mutations Determining PCSK9 Levels

By screening a hypercholesterolemic cohort of individuals lacking mutations in the LDLR and APOB, FH cases attributed to PCSK9 mutations were estimated at ≈2% of patients with FH. 14,30 However, because the LDLR is the major route for PCSK9 uptake, its circulating levels are higher in patients with FH with LOF LDLR mutations, which may contribute to the wide spectrum of FH. 127 PCSK9 GOF was associated with increased severity of coronary atherosclerosis in patients with polygenic hypercholesterolemia. 128 Plasma PCSK9 levels are increased not only in patients with FH but also in patients with Familial Combined Hypercholesterolemia. 129 These suggest that rare missense mutations in PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations. Similarly, the APOE genotype will influence the lipid phenotype of PCSK9 mutations as suggested in 1 study, with special emphasis on the E3/E2 genotype. 117

Variations With Exceptional Mechanisms

Some mutations allowed a better understanding of the biosynthesis and secretion of PCSK9 biology and are worth highlighting. (1) The French Canadian LOF Q152H mutation prevents the autocatalytic processing of proPCSK9, resulting in a dominant negative form of the protein that in a heterozygote state reduces the circulating levels of PCSK9 and LDL-C by as much as ≈80% and ≈50%, respectively. 22 This mechanism was later confirmed after an exhaustive analysis of all possible Gln152 mutations. 34 (2) Intriguingly the GOF R218S mutation significantly decreases PCSK9 catabolism, allowing it to circulate longer and negatively affect the LDLR to promote high cholesterol. 130 This is likely because of the resistance of this mutant to Furin inactivation. 63 (3) Conversely, the LOF A443T 19 mutation likely results in a novel PCSK9 O-glycosylation site that favors Furin-induced degradation of the protein. 63 (4) The most severe Anglo-Saxon GOF mutation D374Y, 20 which despite lower circulating levels, results in a 10- to 25-fold higher binding affinity of PCSK9 to the LDLR. 36 (5) A few variations in PCSK9 have also been reported in the hinge region (Figure 2 eg, the LOF R434W mutation resulting in lower secretion levels of PCSK9 and reduced circulating LDL-C levels, likely because of a negative effect on the folding of the protein in the ER). 131 (6) On the opposite side, the LDLR GOF H306Y mutation in the EGF-A domain associated with PCSK9-binding results in an enhanced PCSK9-mediated cellular degradation. 132

PCSK9-Based Therapies and Safety Considerations

Companies are racing to develop a drug that mimics the effects of LOF PCSK9 mutations. As previously mentioned, PCSK9 enhances the post-translational degradation of the LDLR, therefore decreasing its capacity to lower LDL-C. This makes PCSK9 a promising therapeutic target 6,30 and several pharmaceutical companies in active clinical or preclinical trials are testing various approaches to inhibit PCSK9. 15,133,134 The best approach to date is the use of a monoclonal antibody (mAb) to PCSK9 that blocks its binding to the LDLR, via an allosteric mechanism. Antibodies targeted against PCSK9 showed remarkable cholesterol lowering in mice and monkeys, where a single injection results in an amazing reduction in LDL-C levels by ≈80% for more than a week. 67

Phase I and II clinical trials in humans have been conducted by many pharmaceutical companies like Sanofi/Regeneron and Amgen phase I trials confirmed safety and efficacy. In phase II trials, both companies report an LDL-C reduction varying between 60% and 70% on subcutaneous injection of ≈140 to 150 mg of the mAb every 2 weeks, with no significant elevation in liver enzymes. 15,133 Interestingly, the combination of 80 mg atorvastatin with a 150 mg PCSK9 mAb had no >7% extra lowering effect on LDL-C achieved by the mAb alone. 135 However, we will have to await the results of long-term administration of a mAb with or without a statin before deciding whether a mAb monotherapy is sufficient. As an added benefit, such mAbs also reduced by ≈30% the levels of the highly atherogenic Lp(a), suggesting that PCSK9 also targets the hepatic Lp(a) receptor(s) for degradation. The 2-fold lower reduction of circulating Lp(a) by a PCSK9 mAb when compared with LDL-C suggests that >1 Lp(a) receptor exists, 136 one of which may be targeted for degradation by PCSK9. The identification of the putative PCSK9-sensitive receptor(s) and the segment(s) within that interacts with PCSK9 will surely have an important effect on our understanding of the Lp(a) and PCSK9 biology. Furthermore, because reduction of Lp(a) is currently limited to the use an antisense oligonucleotides to ApoB mRNA or apheresis, 136 defining the PCSK9-sensisitve Lp(a) receptor will surely improve current antiatherogenic therapies. The mAb that will move to routine clinical application will depend on long-term safety data, ease of administration, and price. 126 Online Table I summarizes some of the results of past and ongoing clinical trials. Although injections are not particularly attractive for lifelong treatment, such an approach would likely be embraced by patients having side effects from current lipid-lowering agents or by high-risk subjects striving to achieve lower LDL-C, as indicated by recent guidelines. 137,138 For example, homozygous patients with FH, for whom initial LDL-C levels starts ≈3 to 4× that of the general population, are usually unable to achieve a 50% reduction on available oral agents and thus require LDL-apheresis, a form of dialysis to eliminate the LDL-C from blood 139 Amazingly, administration of a blocking PCSK9 mAb to homozygote patients with FH with some residual LDLR activity resulted in ≈30% reduction in LDL-C, 140 giving some new hope for these patients that have to often undergo apheresis.

Another approach for inhibiting the PCSK9-LDLR extracellular interaction is to use inhibitory adnectins that are specific for PCSK9. Indeed, Bristol-Myers Squibb and Adnexus are currently investigating the efficacy of such an approach in phase I clinical studies. The isolated adnectins are similar to mAbs (which are ≈150 kDa), but smaller in size (≈12 kDa), and comprise a scaffold of the tenth extracellular human fibronectin type III domain that exposes a PCSK9-binding loop.

Because PCSK9 enhances the degradation of the LDLR by an intracellular and extracellular pathway (Figure 2), 48 and unless the liver mostly uses the extracellular pathway, a therapeutical approach that blocks both processes may be more effective than one inhibiting only the extracellular pathway, as with mAbs. The use of antisense oligonucleotides should abrogate both pathways because it reduces mRNA levels of PCSK9 directly. However, both small 2′-O-methoxyethyl–modified phosphorothioate antisense oligonucleotide 141 and locked nucleic acid (LNA) antisense oligonucleotides 142,143 against PCSK9 had a rough start. Although promising results were obtained in mice and monkeys, both phase I clinical trials had to be terminated (http://rnaitherapeutics.blogspot.ca/2011/10/santaris-terminates-pcsk9.html the modified phosphorothioate antisense oligonucleotide trial was terminated for unknown reasons) likely because of serious side effects. Thus, although antisense LNA, which targets both human and mouse PCSK9 with similar efficiency in reducing PCSK9, 142 kidney toxicity led LNA-targeted PCSK9 treatment to be terminated from further clinical trials. 144

A small interfering RNA (siRNA) clinical trial involving siRNA-targeting PCSK9 has been evaluated in a randomized, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL-C of ≥3·mmol/L or higher. 145 The data showed that at a dose of 0.4 mg/kg, this relatively safe treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein and a mean 40% reduction in LDL-C from baseline relative to placebo. Phase II clinical trials are underway. Although mAbs seem to block close to 100% of free circulating PCSK9, the siRNA approach still left ≈30% PCSK9 in circulation, suggesting limited efficacy of the current siRNA method. Although a direct comparison of this approach with the mAb one is yet to be tested in human, the efficacy of the reduction of LDL-C observed with siRNA (40%) 145 is still not better than the one achieved with mAbs (50%–70%). 15,133 It is thus possible that the intracellular pathway in liver may have a relatively minor contribution to the overall activity of PCSK9 on LDLR.

Other anti-PCSK9 approaches may involve the use of Fc-fusion proteins to either the PCSK9 prosegment, 146 or even the R1 domain of annexin A2. 111 None of the published therapeutic anti-PCSK9 approaches in humans used a small-molecule inhibitor, possibly because of the relative flatness of the surface of interaction of the [PCSK9≡LDLR] complex, and thus the absence of a targetable groove that could accommodate a small molecule inhibitor. 30 However, we will have to await the results of some novel approaches toward the identification and testing of small-molecule inhibitors, including those that block either the PCSK9 interaction with the LDLR using EGF-A mimetics 147 or prevent the autoprocessing of proPCSK9 and the secretion of PCSK9, 6 thus mimicking the mutants lacking either an active site residue, 12 the C672X 34 or the Q152H mutation. 22,34 Such novel approaches may overcome the obstacles to small-molecule therapy, especially if they will result in an orally active inhibitor of liver PCSK9.

Because PCSK9 seems to target a number of LDLR family members for degradation, some of which act as entry receptors for infectious viruses, it is recommended that those individuals harboring a viral infection be carefully monitored for viral titers or excluded from anti–PCSK9-based therapy. Furthermore, in view of the critical importance of PCSK9 in liver regeneration, patients undergoing liver resection should not take anti-PCSK9 medication. In addition, chronic administration of an anti-PCSK9 therapy in patients must be monitored for the possible occurrence of insulin resistance and glucose intolerance. However, it must be noted that the glucose intolerance was to date observed only in genetic mutants with global LOF, including male Pcsk9-knockout mice 108 and humans carrying the R46L variant. 117 The use of a mAb may not have similar effects in human. To date, with the short-term treatment (from 12 weeks to 1 year) with either a mAb or siRNA, no evidence was reported for the development of type II diabetes mellitus. However, the gold standard for testing glucose intolerance using an oral glucose challenge has not yet been reported. Finally, because in mice PCSK9 targets VLDLR, 42,65 apoER2, 42 and CD36, 70 reducing circulating PCSK9 concentrations may enhance the levels of these receptors in adipose and other tissues, which may affect FFA clearance. Methods like waist/hip ratios or imaging techniques would need to be implemented to assess the degree of visceral adiposity that may result from the anti-PCSK9 therapy.

Conclusions

With the discovery of PCSK9 in 2003 the lipid field took a sharp turn, with PCSK9 inhibitors becoming an undeniable therapeutic reality (Figure 3). The molecular basis for PCSK9 action supports a model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A–like domain of the LDLR. This reduces LDLR recycling and downregulates LDLR activity, thereby increasing the levels of LDL-C in the blood. Thus, PCSK9 plays a key role in cholesterol homeostasis. Humans with high levels of PCSK9 or GOF mutations have increased levels of plasma LDL-C and significantly enhanced CVD risk during their lifetime. Humans with low levels of PCSK9 or LOF mutations have reduced levels of plasma LDL-C and significantly lower risk of developing a CVD. In addition, PCSK9 exhibits pleiotropic metabolic effects that need to be further explored. Statin therapy results in an increased plasma PCSK9 levels but lower overall LDL-C levels. This suggests that lowering PCSK9 levels may enhance the efficacy of statins to reduce LDL-C. Animal models have proven invaluable to screen a new drug’s modality because atherosclerosis and vascular calcification are enhanced in transgenic -mice overexpressing PCSK9 and reduced in Pcsk9-knockout mice. In humans, loss of 1 copy of PCSK9 prevents 88% of cardiovascular events in the Atherosclerosis Risk in Communities (ARIC) trial on >3000 individuals followed >15 years. 21 Thus, PCSK9 is a clear target for the development of new lipid-lowering therapies. Pharmacologically induced PCSK9 inhibition efficiently reduces LDL-C levels and improves other lipid parameters, such as ceramides and Lp(a). Monoclonal antibodies are at present the most advanced PCSK9 inhibitors in terms of pharmacological development and clinical response. Long-term studies will establish whether the beneficial effects of PCSK9 inhibition on LDL-C levels directly translate into safe and effective CVD risk reduction. Despite its apparent safety, there are concerns about the inhibition of PCSK9 because we still know little about its global physiological functions. Nevertheless, since the discovery of the LDLR and its importance in hypercholesterolemia and regulation by statins, it has been a long time that a new avenue has come about to reduce substantially cholesterol levels. The realization that a complete LOF PCSK9 mutation and inhibition of plasma PCSK9 result in rock-bottom cholesterol levels suggests that PCSK9 inhibitors could be the next blockbuster drug to combat hypercholesterolemia, which would be a harbinger of things to come. 148

Figure 3. Proprotein convertase subtilisin kexin 9 (PCSK9): from discovery to clinic. The pace of research, from PCSK9 discovery through to clinical trials, has been rapid starting from its discovery in 2003 and the proof of principle that monoclonal antibody (mAb) can inhibit its function in 2010, all the way to ongoing phase III clinical trials. CVD indicates cardiovascular disease FH, familial hypercholesterolemia GOF, gain-of-function KO, knockout LDL-C, low-density lipoprotein-cholesterol LDLR, LDL receptor LOF, loss-of-function and NARC, neural apoptosis regulated convertase.


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Introduction

Caenorhabditis elegans is an important model system in biology, because of its tractable size (959 somatic cells in adult hermaphrodites), its genetic manipulability, and its optical transparency, which yields the possibility of whole-organism imaging of biological processes and signals. Perhaps not surprisingly, therefore, super-resolution microscopy has been useful to the analysis of C. elegans, with studies applying STORM, PALM, SR-SIM, and STED to C. elegans to investigate cells and tissues in both intact or dissected C. elegans (Rankin et al., 2011 Gao et al., 2012 Vangindertael et al., 2015 He et al., 2016 Köhler et al., 2017 Krieg et al., 2017). However, the depths of imaging of such studies were largely physically limited to a few microns to tens of microns, insufficient to map the entire depth of an adult animal, and the hardware required for super-resolution microscopy is not available in all laboratories, and can be slow and/or expensive to deploy. Furthermore, the tough cuticle of C. elegans presents a barrier to immunostaining in the intact animal, important for STORM and STED imaging and for the general labeling of proteins in a variety of scientific contexts.

Recently, we discovered that it is possible to isotropically expand biological specimens by permeating them evenly and densely with a swellable hydrogel polymer network, anchoring key biomolecules or labels to the hydrogel, softening the tissue through a chemical process, and then adding water, which swells the polymer and in turn the tissue (Chen et al., 2015). This technique, expansion microscopy (ExM), is now being adapted and improved by many groups, and has been applied to tissues of mice, human patients, and in many other biological contexts (Chen et al., 2015 Chen et al., 2016 Chozinski et al., 2016 Ku et al., 2016 Tillberg et al., 2016 Chang et al., 2017 Zhao et al., 2017 Park, 2018 Truckenbrodt et al., 2018 Gambarotto et al., 2019 Wassie et al., 2019). However, C. elegans is wrapped in a multi-layer cuticle, which is well known to be impermeable to many small molecules and all antibodies, and mechanically stiff to the point where physical expansion would be expected to proceed poorly (Duerr, 2006 Page and Johnstone, 2007 Chisholm and Xu, 2012). Thus, we set out to develop an ExM protocol customized for the C. elegans context that would overcome these barriers.

To achieve this goal, we modified previously published protocols in a number of ways (Figure 1, green steps) to generate a new protocol which we call expansion of C. elegans (or ExCel). This protocol results in high signal-to-background antibody staining against protease-resistant fluorescent proteins, low-distortion (

1–6% over length scales of 0–100 μm) physical expansion by

3.3x, and both protein and RNA detection with sub-cellular resolution. Using ExCel, we were able to resolve synaptic and gap junction proteins better than with ordinary confocal microscopy, and simultaneously image proteins, RNA, and DNA location within the same specimen. In particular, such multiplexed capability has not been demonstrated with previous super-resolution methods in C. elegans, and facilitates nanoscale-precise analyses of how multiple molecular types are spatially organized in the context of an entire animal.

Workflow for expansion of C. elegans (ExCel) sample processing.

A method for expanding cuticle-enclosed intact C. elegans, extending published proExM and ExFISH protocols with specific modifications (shown in green text full key in lower left). Depending on whether the user intends to visualize RNAs or not, the protocol branches into two forms. The protocol without ExFISH, which supports the readout of fluorescent proteins, DNA location (in the form of DAPI staining), and anatomical features, is indicated with blue arrows, ending in Panel L. The protocol with ExFISH, which additionally supports readout of RNAs, is indicated with orange arrows, ending in panel Q. For all steps after hydrogel formation (Panels G-Q), the linear expansion factor of the hydrogel-specimen composite is shown in parentheses. (A–Q) Steps of the protocol, with the bold text indicating the title of the step see text for details of each step.

The standard ExCel protocol visualizes fluorescent reporters, such as those fused to proteins of interest, which requires transgenesis, and could in principle affect the function and localization of the target protein. Thus, we additionally developed an alternative ExCel protocol, which we call epitope-preserving ExCel, that enables detection of untagged, completely endogenous proteins, using off-the-shelf primary antibodies. The epitope-preserving ExCel protocol replaces the use of Proteinase K, a general protease that disrupts most epitopes in the standard ExCel protocol, with an epitope-preserving cuticle-permeabilization treatment that we identified in a systematic screen of chemical treatments. This protocol enables antibody staining of protein epitopes at the expense of a slightly reduced expansion factor (

2.8x) and lower expansion isotropy (

8–25% error over length scales of 0–100 μm). We showed that epitope-preserving ExCel allows multiplexed readout of multiple native proteins at super-resolution, a capability that we used to identify a previously unreported protein localization at the junctions between developing vulval precursor cells, and to resolve the peri-active and active zones of chemical pre-synapses.

Lastly, we developed a third protocol, iterative ExCel (iExCel), which enables two successive rounds of hydrogel-mediated expansion of a given worm, by incorporating the previously validated strategies of iterative expansion microscopy into the ExCel context (Chang et al., 2017). iExCel brings the expansion factor from

20x, and the theoretical limit of resolution down to

25 nm, at a low level of distortion (

1.5–4.5% over length scales of 0–100 μm), on par with that of standard ExCel, on which it builds. With iExCel, we were able to resolve fluorescent puncta that may represent individual GFP molecules expressed in the neuronal cytosol.

Each of these ExCel protocols highlights some of the challenges remaining in deploying ExM in C. elegans, including distortion in the gonad and mouth regions, reduced general isotropy with epitope-preserving ExCel, and the ability to only detect fluorescent proteins with the current form of iExCel, which provide grounds for further optimization in the future.


Abstract

The regulatory effect of growth hormone (GH) on its target cells is mediated via the GH receptor (GHR). GH binding to the GHR results in the formation of a GH-(GHR)2 complex and the initiation of signal transduction cascades via the activation of the tyrosine kinase JAK2. Subsequent endocytosis and transport to the lysosome of the ligand-receptor complex is regulated via the ubiquitin system and requires the presence of an intact ubiquitin-dependent endocytosis (UbE) motif in the cytosolic tail of the GHR. Recently, the model of ligand-induced receptor dimerization has been challenged. In this study, ligand-independent GHR dimerization is demonstrated in the endoplasmic reticulum and at the cell surface by coimmunoprecipitation of an epitope-tagged truncated GHR with wild-type GHR. In addition, evidence is provided that the extracellular domain of the GHR is not required to maintain this interaction. Internalization of a chimeric receptor, which fails to dimerize, is independent of an intact UbE-motif. Therefore, we postulate that dimerization of GHR molecules is required for ubiquitin system-dependent endocytosis.

Postnatal growth as well as lipid and carbohydrate metabolism is regulated by growth hormone (GH). GH effects are mediated by means of the GH receptor (GHR), a type I transmembrane glycoprotein that belongs to the cytokine receptor superfamily. In addition to GHR, this family includes the receptors for erythropoietin (Epo), prolactin, thrombopoietin, leptin, ciliary neurotrophic factor, leukemia inhibitory factor, granulocyte colony-stimulating factor, and several of the ILs (1). Although the overall homology between members is limited, some conserved motifs have been identified (reviewed in ref. 2). Their extracellular domain contains pairs of disulfide-linked cysteine residues and a WSXWS (tryptophan, serine, any amino acid, tryptophan, serine) motif, which is indirectly involved in ligand binding (3). All cytokine receptors lack intrinsic kinase activity. Instead, a conserved proline-rich domain in their cytosolic tail, box 1, functions as a binding site for members of the JAK tyrosine kinase family. In case of GHR, ligand binding results in the activation of JAK2 molecules (4), which in turn phosphorylate tyrosine residues in the receptor cytosolic tail and down-stream signaling molecules (reviewed in ref. 5). The ligand-receptor complex is internalized via clathrin-coated vesicles and subsequently transported via endosomes to lysosomes (6𠄸). We have shown that both endocytosis and transport to lysosomes require an active ubiquitin-conjugation system and can be inhibited by proteasome inhibitors (9�). In particular, a 10-amino acid motif [ubiquitin-dependent endocytosis (UbE)-motif, DSWVEFIELD] in the cytosolic tail of the GHR is essential because mutations here, for instance F327A, inhibit GHR ubiquitination, internalization, and degradation (10, 12).

Based on crystallographic data of GH bound to GHR extracellular domains, it has been postulated that a single GH molecule dimerizes two GHR molecules after which signaling is initiated (3, 13). However, dimerization itself is not sufficient for signal transduction because administration of mAbs directed to the extracellular domain of the GHR resulted in dimerized GHRs but failed to induce signal transduction (14). The GHR extracellular domain contains two subdomains, which are separated by a hinge region (3). With an Ab raised against this hinge region, Mellado et al. (15) demonstrated a conformational change after ligand binding. Recognition of the GHR by this Ab improved after GH binding but not when the GH antagonist GH(G120R), with only one intact GHR binding site, was used (15). Recently, Ross et al. (16) used the mAb Mab5, which was postulated to interfere with GHR dimerization, and demonstrated an increase in the number of binding sites for GH as well as GH antagonist B2036, which also contains one defective GHR binding site. It suggests that B2036 binds to a preformed receptor dimer (16). More evidence was provided by crosslinking studies with 125 I-labeled GH or GH antagonist. Complexes similar in size and corresponding to a ligand:GHR stoichiometry of 1:2 were detected for both ligands (17, 18). Taken together, these data predict the presence of preformed GHR dimers at the cell surface as was also recently proposed for the Epo receptor (EpoR) based on crystallographic data (19, 20) and Ab-mediated immunofluorescence copatching of epitope-tagged EpoRs (21).

Here, we show that GHR is dimerized in the absence of ligand in the endoplasmic reticulum (ER) and at the cell surface. Our data suggest that the extracellular domain of the GHR is not essential for the maintenance of this interaction. Internalization of a chimeric protein, which fails to dimerize, is independent of an intact UbE-motif, suggesting that dimerization is a prerequisite for ubiquitin system-dependent endocytosis.


EMERGING ROLES FOR CYSTEINE PROTEASES IN HUMAN BIOLOGY

AbstractCysteine proteases have traditionally been viewed as lysosomal mediators of terminal protein degradation. However, recent findings refute this limited view and suggest a more expanded role for cysteine proteases in human biology. Several newly discovered members of this enzyme class are regulated proteases with limited tissue expression, which implies specific roles in cellular physiology. These roles appear to include apoptosis, MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development. The ability of macrophages and other cells to mobilize elastolytic cysteine proteases to their surfaces under specialized conditions may also lead to accelerated collagen and elastin degradation at sites of inflammation in diseases such as atherosclerosis and emphysema. The development of inhibitors of specific cysteine proteases promises to provide new drugs for modifying immunity, osteoporosis, and chronic inflammation.


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