Pro-protein convertase subtilisin/kexin type 9 (PCSK9) the ninth known member of the mammalian serine proprotein convertase (PC) family, and plays an important role in low density lipoproteins (LDL) metabolism. Once secreted, PCSK9 binds LDL receptors (LDLRs), targeting them toward intracellular degradation through an endosomal/lysosomal route. Inhibition of PCSK9 can reduce LDLRs degradation and increase the expression of LDLRs in the cell surface, resulting in an enhanced recycling of LDLRs and a reduction in the levels of LDL cholesterol. Hence, inhibitors of PCSK9 suppose a promising therapeutic strategy for the treatment of hypercholesterolemia.

Discovery of PCSK9

PCSK9 was first described as neural apoptosis-regulated convertase 1 (NARC-1) in studies of cerebral neuron apoptosis, suggesting that it could be implicated in the differentiation of cortical neurons ^[1]. Concomitant and following studies in patients with familiar hypercholesterolemia revealed the clinical importance of PCSK9, showing that patients with gain-of-function mutations presented increased levels of cholesterol in plasma (i.e. hypercholesterolemia) due to reduced expression of LDLRs. In contrast, loss-of-function variants of PSCK9 are associated with a reduction of LDL cholesterol levels and a lower risk of cardiovascular disease. The role of PSCK9 in LDLRs and cholesterol metabolism has been confirmed in animal models. Thus, mice overexpressing PCSK9 show a reduction in the expression of hepatic LDLRs and hypercholesterolemia, whereas knockout mice for PCSK9 present decreased levels of plasmatic LDL cholesterol because of increased expression of LDLRs ^[2][3].

Gene and synthesis of PCSK9

Under normal conditions, PCSK9 has a half-life in plasma of approximately 5 minutes. PCSK9 is the ninth known member of the mammalian subtilisin (S8) serine proprotein convertase (PC) family that carries out the proteolytic maturation of secretory proteins such as neuropeptides, prohormones and cytokines. Humans have nine different PCs that can be divided between S8A and S8B subfamilies. PCSK9 is classified in subfamily S8A ^[4].

The human gene for PCSK9 is 22 kb length and it is located in chromosome 1p32.3. It contains 11 introns and 12 exons that encode the 692 amino acids of the enzyme. The sequence of the protein is characterized by a signal sequence (amino acids 1-30), a prodomain (amino acids 31-152), and a catalytic domain, followed by a C-terminal region of 243 amino acids which is rich in cysteine and histidine residues Fig. 1. PCSK9 is mainly expressed in the liver, intestine and kidney, and it can also be in the nervous system. It is synthesized as a precursor of ~74 kDa that is processed in the endoplasmic reticulum (ER) where it undergoes cleavage of its signal peptide and intramolecular autocatalytic cleavage producing a ~60-kDa catalytic fragment. The autocatalysis of the zymogen takes place between Gln152 and Ser153 ^[5]. This cleavage is necessary for transport from ER to the Golgi body and for secretion. The cleaved prodomain of ~14 kDa remains associated with the catalytic domain, which is unique to PCSK9. This facilitates protein folding, permits the mature protein to move from ER into the secretory pathway and regulates the catalytic activity of the enzyme by blocking the access to the catalytic site Fig. 2? ^[6][7].

PCSK9 can be found in plasma in two forms: the mature and secreted form of ~60 kDa, and as an inactivated fragment of ~53 kDa produced by the cleavage of the mature form at the motive RFHR218↓ by other proprotein convertases, mainly furin and/or PC5/6A ^[8].

In humans, PCSK9 circulates in plasma in a phosphorylated state and it has been shown that it is phosphorylated at the Ser47 and Ser688 by a Golgi casein kinase-like kinase ex vivo. This phosphorylation might be important to protect the propeptide against proteolysis ^[9].

Binding to LDLR

Yamamoto et al., described a two-step model wherein the Pro-Cat domain of PCSK9 initiates contact with EGF-A of the LDL receptor at neutral pH. An antiparallel β-sheet is formed between residues 377– 379 of PCSK9 and residues 308–310 of EGF-A. The complex PCSK9:LDLR is internalized and exposure to the low pH environment of the endosome, where the CT domain of PCSK9 binds the Ligand-Binding domain of LDLR. This interaction impair the ability of the receptor to adopt a recycling-competent conformation and promote trafficking of the PCSK9-LDLR complex to the lysosome (Yamamoto, Lu et al. 2011).

In the absence of PCSK9, lipoprotein binding to the LDLR leads to receptor-mediated endocytosis. The low pH environment of the endosome induces a conformational change in the LDLR, resulting in discharge of bound lipoprotein ligand and interaction between the β-propeller segment and ligand-binding repeats 4 and 5. This event permits the segregation and separate trafficking of the LDLR to the cell surface and the lipoprotein ligand to the lysosome, respectively (Yamamoto, Lu et al. 2011).

It is believed that the transition from neutral pH at the cell surface to low pH in the endosomal compartment activates a “histidine switch” that promotes the mentioned intramolecular interaction between receptor domains. A critical aspect of this conformational change is that it promotes ligand release, thereby facilitating receptor recycling to the cell surface, where it is available for another round of endocytosis. PCSK9-mediated interference with this process causes the LDLR to traffic to lysosomes, where it is degraded (Yamamoto, Lu et al. 2011).

Kinetics of PCSK9

Under normal conditions, PCSK9 has a half-life in plasma of approximately 5 minutes. It has been showed that in humans and mice, LDLR is a major regulator for PCSK9 levels and clearance, therefore in the presence of an additional copy of LDLR in the liver (induced by transgenic expression) reduces the half-life of PCSK9 by 50%, to 2.9 minutes, whereas in the absence of LDLR, the half-life of PCSK9 in serum is prolonged between 3–10 times above normal.

The kinetics of wild-type (WT) PCSK9 binding to LDLR shows Kd(poner d pequeña) values that range from 90 to 840 nM at neutral pH, and its affinity to LDLR becomes ∼100-fold higher at lower pH with Kd(poner d pequeña) values ranging from 1–8 nM. (Relacionar con los cambios estructurales de arriba)

PCSK9 binding to LDLR has been described as biphasic, with a first rapid phase characterized by a half-time of 6.6 minutes, which accounts for 35% of the equilibrium binding and a second slow phase whose half-time is 94 minutes. Similarly, 25% of the PCSK9 bound to LDLR dissociates during the rapid phase with a half-time of 19 minutes, while the remaining PCSK9 dissociates slowly with a half-time of 297 minutes.

Despite the rapid binding of PCSK9 and internalization of LDLR by hepatocytes, PCSK9-mediated degradation of LDLR in vitro has only been observed after several hours. It was further shown that, at least in mice, PCSK9 remains intact in the liver for up to 4 hours after its internalization, thus suggesting that other events might be required in order to allow PCSK9-mediated degradation of LDLR (or LDLR mediated degradation of PCSK9).

(Giunzioni and Tavori 2015)

You may include any references to papers as in: the use of JSmol in Proteopedia ^[10] or to the article describing Jmol ^[11] to the rescue.

PCSK9 as a therapeutic target

Regarding the fact that PCSK9 reduces the LDL-R and thus LDL-C clearance from blood, high concentrations of this protein in plasma increase risk to suffer from cardiovascular diseases. Effect that has been considerably reversed when studying loss of function mutations in PCSK9 which conditioned self-processing and secretion. Hence, several studies approaching alternative treatments against hypercholesterolemia using PCSK9 as new target to avoid side effects of statins treatment. To this end inhibitory strategies against PCSK9 are under investigation with different approaches that can either prevent the protease from binding LDL-R or inhibiting its synthesis and processing.

Extracellular inhibitors

Their strategy is focused in the reduction of PCSK9 function or its plasma level. The two main inhibitors are:

Monoclonal antibodies

They constitute the most successful strategy via sequestrating in plasma circulating PSCK9 binding to a specific epitope in the molecule. By binding to the catalytic domain and prodomain of the protease they neutralize PCSK9 activity, thus, preventing its interaction with LDL-R. In clinical trials they reached a maximum a suppression of plasma free PCSK9 after 4 to 8 hours of administration achieving a 65% reduction of LDL-C in healthy patients and a 60 to 80% reduction in patients with hypercholesterolemia. There are three known mAb that have reached the clinical trials evolocumab, alirocumab and bococizumab. Only the two first antibodies are fully human while the last one is approximately 3% murine which has been withdrawn due to anti-drug antibodies responses. Out of metanalyses it has been addressed that they reduce cardiovascular mortality as well as the rate of myocardial infarction. Both alirocumab (Praluent®) and evolocumab (Repatha®) received FDA and EMA approval and are indicated as complement to diet and maximally tolerated therapy for the treatment of adults with heterozygous familial hypercholesterolemia or clinical atherosclerotic CVD requiring additional lowering of LDL-C.

Pharmacodynamics: Antibodies interaction with PCSK9 is based in EGFA binding site of the peptidase. In affinity studies, unravelling of the mechanism of interaction of antibodies with PCSK9 was carried out using an antibody phage library. Among them, the one which most potently inhibited PCSK9 interaction with LDLR was antibody 33 (Fab33) also known as RG7652 causing a reduction of LDL-C levels in humans. Its epitope is centered on EGFA binding site and the antibody engages to it by 5 (H1, H2, H3, L1, L3) of its 6 complementary-determining region (CDR) loops. As well an additional hydrogen bond is formed by residues near the heavy chain residue 73. An approximated 950 Å2 surface area is buried at each side Fab33-PCSK9 contact with 73% of this area buried by the heavy chain. Thus, mechanism underlying interaction is based in the CDR-H2 loop of the Fab33 which is projected to the N terminal groove of PCSK9 which is normally occupied by P´ helix. Consequently P'helix, which by its P1' Ser 153 and P3' Pro 155 residues stabilize the bound of PCSK9 to LDLR-EGFA domain via polar and Van der Waal interactions, is displaced and cleaved (In downstream P'helix Arg165-Tyr166 residues).

Disease

Relevance

Structural highlights

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