Subtilisin Kexinlike Isozyme1 SK1

Natural Cholesterol Guide

Beat Cholesterol Guide

Get Instant Access

In 1999, using reverse transcriptase-PCR and degenerate oligonucleotides, derived from the active-site residues of subtilisin/kexin-like serine proteinases, we identified in human, rat, and mouse, a type I membrane-bound proteinase, which we called subtilisin/kexin-isozyme-1 (SKI-1) [58]. It was so named because of the homology of its catalytic domain to the bacterial subtilisin BPN (Figure 4). In contrast to the basic amino acid-specific PCs, this convertase appears to prefer processing precursors at residues within the general motif RX(V, I,L)(K,F,L)j, with the preferred critical basic Arg/Lys and aliphatic (Leu/Ile/Val) residues occupying positions P4 and P2, respectively [58].

Data bank searches revealed that Sakai et al., also identified a few month earlier a similar hamster enzyme from CHO cells, which they named Site-1 protease (S1P). They determined that this enzyme was involved in the control of lipid metabolism by mediating the cleavage of Sterol Regulatory Element-Binding Proteins (SREBPs) in its luminal loop [59]. Previously, SREBPs were described to play a key role in the fundamental feedback mechanism of cellular lipid homeostasis.

The transcriptional activation of genes containing sterol responsive elements (SRE) is known to be regulated by sterols through modulation of the proteolytic maturation of SREBPs [59]. The two known SREBPs (SREBP1 and SREBP2) are inserted into the membrane of the endoplasmic reticulum envelope in a wide variety of tissues. In sterol-deficient cells, proteolytic cleavage of SREBPs by SKI-1 and S2-P protease releases their N-terminal mature form from the membrane into the cytosol enabling them to enter the nucleus (Figure 5), where they bind to the SREs and activate genes involved in the biosynthesis of cholesterol, triglycerides, and fatty acids [59]. In the presence of sterols, the proteolytic process is inhibited and the transcription of the genes is reduced [59] (Figure 5).

The gene of SKI-1/S1P (PCSK8) is located on human chromosome 16 and mouse chromosome 8 (Figure 3), and is expressed in most tissues and cells. To date, several viral glycoproteins in addition to SREBPs, as well as the brain-derived

Signal peptide :_: Pro-segment » Catalytic domain _ P domain

GF-cytokine-R Transmembrane domain jgg Cytoplasmic domain

Signal peptide :_: Pro-segment » Catalytic domain _ P domain

GF-cytokine-R Transmembrane domain jgg Cytoplasmic domain

Figure 4. Schematic representation of the prohormone convertases SK-1 and NARC-1. The convertase subtilisin/kexin-isozyme-1 (SKI-1) possesses a catalytic domain with high homology to bacterial subtilisin BPN, whereas the neural apoptosis-regulated convertase-1 (NARC-1) belongs to the proteinase K-like subtilases neurotrophic factor, ATF-6 and endocrine polypeptide somatostatin were found to be SKI-1 substrates [59-64]. New substrates include CREB-containing precursors, such as CREB-4 were also reported to be cleaved by SKI-1/S1P [65]. As with the PCs, the precursor protein of SKI-1 is also autocatalyticaly cleaved (Figure 2) and can be further processed into two membrane-bound forms of SKI-1 (120 and 106 kDa), differing by the nature of their N-glycosylation. Some of these SKI-1 forms are shed into the medium as a 98-kDa form.

2.2 Neural Apoptosis-regulated Convertase 1 (NARC-1/PCSK9)

Through a search of patent databases, using as a bait a small sequence of the conserved catalytic domain of SKI-1/S1P, we identified a protein belonging to proteinase K-like subtilases (Figure 4) called neural apoptosis-regulated convertase 1 (NARC-1) or PCSK9. NARC-1/PCSK9 was previously identified by two pharmaceutical companies [66], based on the cloning of up-regulated cDNAs after the induction of apoptosis by serum deprivation in the primary cerebellar neurons and by means of global cloning of secretory proteins [66]. Like other convertases, NARC-1/PCSK9 is also synthesized as a zymogen that undergoes autocatalytic intramolecular processing in the ER (Figure 2). This cleavage occurs within the

Figure 5. Role of SKI-1/S1P in the processing of SREBP. The sterol regulatory element binding protein precursors (SREBPs) are inserted into the membrane of the endoplasmic reticulum (ER) envelope in various tissues and the amino-terminal transcription-factor domain (bHLH-zip) is located in the cytoplasmic compartment. Under insufficient amount of sterols, the SREBP precursor protein travels to the Golgi apparatus where SKI-1/S1P cleaves at site-1 in the luminal loop and produce the substrate for the Site-2 protease (S2P), which cleaves at site-2. This second cleavage releases the transcription-factor domain from the membrane that enters the nucleus and induces the increased transcription of target genes. In the presence of sterols, the proteolytic process is inhibited and the transcription of the genes is reduced. bHLH-zip: basic helix-loop-helix leucine-zipper

Figure 5. Role of SKI-1/S1P in the processing of SREBP. The sterol regulatory element binding protein precursors (SREBPs) are inserted into the membrane of the endoplasmic reticulum (ER) envelope in various tissues and the amino-terminal transcription-factor domain (bHLH-zip) is located in the cytoplasmic compartment. Under insufficient amount of sterols, the SREBP precursor protein travels to the Golgi apparatus where SKI-1/S1P cleaves at site-1 in the luminal loop and produce the substrate for the Site-2 protease (S2P), which cleaves at site-2. This second cleavage releases the transcription-factor domain from the membrane that enters the nucleus and induces the increased transcription of target genes. In the presence of sterols, the proteolytic process is inhibited and the transcription of the genes is reduced. bHLH-zip: basic helix-loop-helix leucine-zipper motif SSVFAQ SIP [67]. Northern blots and in situ hybridization analyses revealed that in the adult NARC-1/PCSK9 mRNA expression is restricted to the liver, kidney and small intestine. Unlike PC7 and SKI-1, but similar to Furin, PC5 and PACE4, the mRNA of NARC-1/PCSK9 was up-regulated during liver regeneration following partial hepatectomy [68]. Overexpression of NARC-1/PCSK9 in primary culture of embryonic telencephalon cells at day 13.5 induced differentiation of neuronal progenitors, suggesting a role for NARC-1/PCSK9 in enhancing the differentiation/proliferation of cortical neurons [66]. Recently, we have shown that point mutations in human PCSK9 are associated with the development of severe hypercholesterolemia phenotypes [69], likely through a grain of function [70]. Conversely, other mutations resulting in early termination of the coding region (non-sense mutations) resulted in a loss of function and hence familial hypocholes-terolemia [71]. Thus, mutations in PCSK9 results in a dominant form of either hypo-or hyper-cholesterolemia, suggesting that inhibitors of these enzymes may lead to novel pharmaceutical drugs to further lower circulating cholesterol levels as a supplement to the conventional HMG-CoA reductase inhibitors known as "statins".

Was this article helpful?

0 0
Lower Your Cholesterol In Just 33 Days

Lower Your Cholesterol In Just 33 Days

Discover secrets, myths, truths, lies and strategies for dealing effectively with cholesterol, now and forever! Uncover techniques, remedies and alternative for lowering your cholesterol quickly and significantly in just ONE MONTH! Find insights into the screenings, meanings and numbers involved in lowering cholesterol and the implications, consideration it has for your lifestyle and future!

Get My Free Ebook


Post a comment