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PubMed1994

Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases.

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca(2+)-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2', S3, S5 and S6 subsites may also contribute to substrate binding. Based on this model, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.

Amino Acid Sequence↗

PubMed2003

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site, 2: relevance of an N-terminal helix.

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Amino Acid Sequence↗

PubMed2001

Proprotein convertase PACE4 is down-regulated by the basic helix-loop-helix transcription factor hASH-1 and MASH-1.

PACE4 is a mammalian subtilisin-like proprotein convertase that activates transforming growth factor (TGF)-beta-related proteins such as bone morphogenetic protein 2 (BMP2), BMP4 and Nodal and exhibits a dynamic expression pattern during embryogenesis. We recently determined that the 1 kb 5'-upstream region of the PACE4 gene contains 12 E-box (E1-E12) elements and that an E-box cluster (E4-E9) acts as a negative regulator [Tsuji, Yoshida, Hasegawa, Bando, Yoshida, Koide, Mori and Matsuda (1999) J. Biochem. (Tokyo) 126, 494-502]. It is known that the mammalian achaete-scute homologue 1 (MASH-1) binds specifically to an E-box (CACCTG) sequence in collaboration with E47, a ubiquitously expressed basic helix-loop-helix (bHLH) factor. To identify the roles of the bHLH factor and E-box elements in regulating PACE4 gene expression in neural development, we analysed the effects of human achaete-scute homologue 1 (hASH-1) on PACE4 gene expression with various neuroblastoma cell lines. The expressions of PACE4 and hASH-1 are correlated inversely in these cell lines. The overexpression of hASH-1 or MASH-1 causes a marked decrease in endogenous PACE4 gene expression but has no effect on the expression of other subtilisin-like proprotein convertases such as furin, PC5/6 and PC7/8. In contrast, other neural bHLH factors (MATH-1, MATH-2, neurogenin 1, neurogenin 2, neurogenin 3 and E47) did not affect PACE4 gene expression. Furthermore, an E-box cluster was a negative regulatory element for the promoter activity in NBL-S cells expressing hASH-1 at high level as determined by a luciferase assay. Binding of hASH-1 to the E-box cluster was confirmed by gel mobility-shift assay. In the present study we identified the PACE4 gene as one of the targets of hASH-1, which is a key factor in the initiation of neural differentiation. These results suggest that the alteration of PACE4 gene expression by hASH-1 causes rapid changes in the biological activities of TGF-beta-related proteins via post-translational modification of these proteins.

Basic Helix-Loop-Helix Proteins↗

PubMed2004

Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase.

OBJECTIVE: Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-beta1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-beta1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-beta1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-beta1 and furin to CFBs MMP-activity and motility. METHODS AND RESULTS: Stimulation of CFBs from adult Sprague-Dawley rats with TGF-beta1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-beta1 activation, indicating that TGF-beta1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-beta1 (20 ng/ml, 24 h) increased their migration by about two-fold (p<0.05), which was accompanied by an enhanced expression and activity of MT1-MMP and MMP-2. Brefeldin A (BFA), a Golgi-disturbing agent, inhibited MT1-MMP activation, indicating that it occurs in the trans-Golgi network (TGN), where furin is concentrated and colocalized with MT1-MMP. Inhibition of furin significantly inhibited TGF-beta1-induced MT1-MMP/MMP-2 activation. Furthermore, inhibition of furin attenuated TGF-beta1-enhanced migration on gelatin-coated membranes (p<0.05). This was comparable to the effects of the MMP-inhibitor GM6001, pointing out that MMPs are major mediators of TGF-beta1-enhanced CFB motility. CONCLUSION: We demonstrate that TGF-beta1 induces MMP-activity in CFBs, thereby facilitating CFBs motility. Furthermore, TGF-beta1 amplifies its activating convertase furin, which is also required for MT1-MMP/MMP-2 activation in CFBs. Thus, furin is central for TGF-beta1 and MT1-MMP activation and might be a novel target in cardiac remodeling.

Animals↗

PubMed2003

Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type.

Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+). Though the chain of events initiating FAF has been elucidated recently, uncertainty remains about the mechanistic details that allow the FAF variants to be processed. To test the hypothesis that impaired Ca(2+) binding in the D187 variants, but not other factors specific to residue 187, increases susceptibility to aberrant proteolysis and subsequent amyloidogenesis, we designed the gelsolin variant E209Q to remove a different Ca(2+) ligand from the same Ca(2+) site that is affected in the FAF variants. Here, we show that E209Q domain 2 does not bind Ca(2+) and is not stabilized against denaturation or furin proteolysis, analogous to the behavior exhibited by the FAF variants. Transfection of full-length E209Q into COS cells results in secretion of both the full-length and furin-processed fragments, as observed with D187N and D187Y. Mutation of the furin consensus sequence in D187N and E209Q gelsolin prevents cleavage during secretion, indicating that inhibition of proprotein convertases (furin) represents a viable therapeutic approach for the treatment of FAF. Mutations that diminish domain 2 Ca(2+) binding allow furin access to an otherwise protected cleavage site, initiating the proteolytic cascade that leads to gelsolin amyloidogenesis and FAF.

Amyloid Neuropathies, Familial↗

PubMed2001

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization.

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.

Amyloidosis, Familial↗

PubMed2026

Biological Mechanisms Underlying the Cardiovascular Effects of Branched-Chain Amino Acids: A Proteome-Wide Mendelian Randomization Study.

BACKGROUND: Ischemic heart disease (IHD) is the leading cause of morbidity and mortality. Branched-chain amino acids (BCAAs) are associated with higher IHD risk, but the underlying biological pathways remain unclear. OBJECTIVES: This study aims to explore these pathways using 2-step proteome-wide Mendelian randomization. METHODS: We examined the associations between genetic proxies for BCAAs and 2922 proteins in the United Kingdom Biobank Pharma Proteomics Project, supplemented by a meta-analysis with data from deCODE to identify proteins associated with BCAAs. Next, we tested their effects on IHD risk using Coronary Artery Disease Genome-wide Replication and Meta-analysis plus Coronary Artery Disease Genetics Consortium (122,733 cases and 424,528 controls) and replicated in FinnGen (31,640 cases and 187,152 controls). We conducted sensitivity analyses using genetic instruments from deCODE. Proteins associated with IHD risk and, in a consistent direction, with genetically predicted BCAAs were considered potential mediators. RESULTS: Genetic proxies for BCAAs were associated with 40 proteins. Among these, 6 proteins showed consistent evidence of mediation, including complement C1s subcomponent, coagulation factor II, granulin, proprotein convertase subtilisin/kexin type 9, sex hormone-binding globulin, and V-set and transmembrane domain-containing protein 2-like. These proteins are involved in inflammation, coagulation, lipid metabolism, and cellular stress response. All associations were robust across different analytical methods and replicated in independent datasets. Mediation analysis showed that these proteins accounted for 6.5% to 32.1% of the association between BCAAs and IHD risk. CONCLUSIONS: This study identified 6 proteins that potentially link BCAAs to IHD, implicating pathways related to inflammation, coagulation, lipid metabolism, and cellular stress responses. To our knowledge, these findings provide novel mechanistic insights into the BCAA-IHD relationship and highlight potential protein targets for future prevention and intervention strategies.

Amino Acids, Branched-Chain↗

PubMed2002

Protein engineering and properties of human metalloproteinase and thrombospondin 1.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

ADAM Proteins↗

PubMed2026

Association of PCSK9 and CCL22 gene polymorphisms with myocardial infarction in a South Indian population.

Myocardial infarction (MI) remains a major global cause of morbidity and mortality, with a particularly high burden among individuals with type 2 diabetes mellitus (T2DM). Host genetic factors play a significant role in modulating individual susceptibility to MI by influencing lipid metabolism and immune-mediated inflammatory pathways. The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is a key regulator of cholesterol homeostasis, while C-C motif chemokine ligand 22 (CCL22) is involved in immune cell recruitment and vascular inflammation. In this study, we investigated the association of PCSK9 rs505151 and rs11591147 and CCL22 rs4359426 polymorphisms with MI risk in a South Indian population. This case-control study included 400 participants categorized into controls (n = 100), MI (n = 100), T2DM (n = 100), and MI with T2DM (n = 100). Significant differences in clinical and biochemical parameters, including lipid indices and cardiometabolic risk markers, were observed between groups (p < 0.05). Genetic analysis revealed a significant association between the PCSK9 rs505151 variant and MI susceptibility across allelic and genotypic distributions, with significant effects under dominant and recessive inheritance models. Multivariable logistic regression confirmed that the rs505151 risk genotype was independently associated with MI after adjustment for age, sex, body mass index, and smoking status. In contrast, PCSK9 rs11591147 was rare and showed no significant association. The CCL22 rs4359426 polymorphism showed limited evidence of association with MI, with a significant effect observed only under the dominant inheritance model. Furthermore, combined analysis using a genetic risk score suggested that cumulative genetic burden involving PCSK9 and CCL22 variants was associated with an increased risk of MI. Overall, our findings suggest that genetic variation in lipid-regulatory and immune-related pathways may contribute to MI susceptibility in South Indians. Further studies are warranted to validate these associations and clarify their biological and clinical relevance.

Humans↗

PubMed2006

The C679X mutation in PCSK9 is present and lowers blood cholesterol in a Southern African population.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) can cause familial hypercholesterolemia. However, two nonsense variants of PCSK9, Y142X and C679X, found in approximately 2% of black American subjects, are associated with a 28% reduction in mean low density lipoprotein (LDL)-cholesterol. We sought to determine the frequency and effect of these nonsense variants in an African population. METHODS AND RESULTS: PCSK9 genotypes were determined in 653 black African women attending two antenatal clinics in Zimbabwe. C679X occurred in 3.7% of subjects and was associated with a 27% reduction in LDL-cholesterol (1.6+/-0.3 mmol/L versus 2.2+/-0.7 mmol/L in non-carriers). We did not observe the Y142X variant. CONCLUSIONS: Our results show that the PCSK9 C679X variant has a marked cholesterol-lowering effect.

Adult↗

PubMed1995

Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.

Amino Acid Sequence↗

PubMed1998

Processing of the Ebola virus glycoprotein by the proprotein convertase furin.

In the present study, we have investigated processing and maturation of the envelope glycoprotein (GP) of Ebola virus. When GP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments, the mature form and two different precursors were identified. First, the endoplasmic reticulum form preGPer, full-length GP with oligomannosidic N-glycans, was detected. preGPer (110 kDa) was replaced by the Golgi-specific form preGP (160 kDa), full-length GP containing mature carbohydrates. preGP was finally converted by proteolysis into mature GP1,2, which consisted of two disulfide-linked cleavage products, the amino-terminal 140-kDa fragment GP1, and the carboxyl-terminal 26-kDa fragment GP2. GP1,2 was also identified in Ebola virions. Studies employing site-directed mutagenesis revealed that GP was cleaved at a multibasic amino acid motif located at positions 497 to 501 of the ORF. Cleavage was blocked by a peptidyl chloromethylketone containing such a motif. GP is cleaved by the proprotein convertase furin. This was indicated by the observation that cleavage did not occur when GP was expressed in furin-defective LoVo cells but that it was restored in these cells by vector-expressed furin. The Reston subtype, which differs from all other Ebola viruses by its low human pathogenicity, has a reduced cleavability due to a mutation at the cleavage site. As a result of these observations, it should now be considered that proteolytic processing of GP may be an important determinant for the pathogenicity of Ebola virus.

Animals↗

PubMed2004

Two engineered eglin c mutants potently and selectively inhibiting kexin or furin.

Eglin c with mutants L45R and D42R at the P(1) and P(4) positions has been reported to become a stable inhibitor toward the proprotein convertases (PC), furin and kexin, with a K(i) of 2.3x10(-8) and 1.3x10(-10) M, respectively. The mutant was further engineered at the P(2)'-P(4)' positions to create a more potent and selective inhibitor for each enzyme. The residue Asp at P(1)' which is crucial for stabilizing the conformation of eglin c remained unchanged. The eglin c mutants cloned into the vector pGEX-2T and expressed in Escherichia coli (DH5alpha) were purified to homogeneity, and their inhibitory activities toward the purified recombinant furin and kexin were examined. The results showed that (1) Leu47 at P(2)' replaced with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes; (2) the replacement of Arg with Asp at P(3)' was favorable for inhibiting furin with a K(i) of 7.8 x 10(-9) M, but not for inhibiting kexin; (3) the replacement of Tyr with Glu at P(4)' increased the inhibitory activity to kexin with a K(i) of 3 x 10(-11) M, but was almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site-directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P' positions.

Amino Acid Sequence↗

PubMed2003

Impaired intestinal proglucagon processing in mice lacking prohormone convertase 1.

The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature glucagon in the pancreas. Disruption of PC1 results in dwarfism and also multiple neuroendocrine peptide processing defects. This study compares the pancreatic and intestinal processing of proglucagon in mice lacking PC1 expression with that in age-matched wild-type controls. Because proglucagon was found to precipitate in acidic extracts, the intestinal processing profile was analyzed in both acidic and neutral extracts by gel filtration, HPLC, and RIA. Supporting a central role for PC2 in glucagon biosynthesis, we found normal processing of proglucagon to glucagon in the pancreas, whereas the intestinal proglucagon processing showed marked defects. Tissue proglucagon levels in null mice were elevated, and proglucagon processing to glicentin, oxyntomodulin, and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2) was markedly decreased, indicating that PC1 is essential for the processing of all the intestinal proglucagon cleavage sites. This includes the monobasic site R(77) and, thereby, production of mature, biologically active GLP-1. We also found elevated glucagon levels, suggesting that factors other than PC1 that are capable of processing to mature glucagon are present in the secretory granules of the L cell. These findings strongly suggest that PC1 is essential for intestinal proglucagon processing in vivo and, thereby, plays an important role in production of the incretin hormone GLP-1 and the intestinotrophic hormone GLP-2.

Animals↗

PubMed2004

Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.

OBJECTIVE: Neural apoptosis-regulated convertase (NARC)-1 is the newest member of the proprotein convertase family implicated in the cleavage of a variety of protein precursors. The NARC-1 gene, PCSK9, has been identified recently as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The 2 other known genes implicated in ADH encode the low-density lipoprotein receptor and apolipoprotein B. As an approach toward the elucidation of the physiological role(s) of NARC-1, we studied its transcriptional regulation. METHODS AND RESULTS: Using quantitative RT-PCR, we assessed NARC-1 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that NARC-1 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. NARC-1 mRNA level was increased by cholesterol depletion but insensitive to liver X receptor activation. Human, mouse, and rat PCSK9 promoters contain 2 typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. CONCLUSIONS: PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of SRE-binding protein (SREBP)-2.

Alitretinoin↗

PubMed2000

The proprotein convertases furin and PACE4 play a significant role in tumor progression.

Processing of latent precursor proteins by proprotein convertases (PCs) into their biologically active products is a common mechanism required for many important biologic functions. This process is tightly regulated, leading to the generation of active peptides and proteins including neuropeptides and polypeptide hormones, protein tyrosine phosphatases, growth factors and their receptors, and enzymes including matrix metalloproteases (MMPs). These processing reactions occurs at pairs of basic amino acids. Within the past several years, a novel family of Ca(2+)-dependent serine proteases has been identified, all of which possess homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This family of PCs is currently comprised of fewer than a dozen members, known as furin/paired basic amino-acid-cleaving enzyme (PACE), PC1/PC3, PC2, PC4, PACE4, PC5/PC6, and PC7/PC8/lymphoma proprotein convertase. They share a high degree of amino-acid identity of 50-75% within their catalytic domains. Despite the relatively high degree of homology in the PC family, only PACE4 and furin localize to the same chromosome: mouse chromosome 7 and human chromosome 15. Recent reports have supported a possible functional role for PCs in tumorigenesis. For instance, convertases have been shown to be expressed in various tumor lines and human primary tumors. Furin and PACE4 process stromelysin 3 (MMP-11 or Str-3), an MMP involved in tumor invasion, into its mature, active form. Similarly, a growing family of MMPs, known as membrane-type metalloproteinases (MT-MMPs), and growth factors and adhesion molecules such as E-cadherin show similar amino-acid motifs and thus could be activated by furin and PACE4. These data, taken together with the high expression levels of PACE4 in 50% of murine chemically induced spindle cell tumors, confer to PACE4 and possibly other PCs a possible functional role in the activation of MMPs and consequently in tumor cell invasion and tumor progression. This was further supported by the remarkable enhancement in the invasive ability of the PACE4-transfected murine tumor cell lines. Mol. Carcinog. 28:63-69, 2000.

Animals↗

PubMed1999

Enzymic characterization in vitro of recombinant proprotein convertase PC4.

Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca(2+) concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at approximately 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from V(max)/K(m) measurements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-acid synthetic peptide, YQTLRRRVKR downward arrowSLVVPTD (where downward arrow denotes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptides, namely Ac-PAKSAR downward arrowSVRA (IGF-I(66-75)) and Ac-PAKSER downward arrowDVST (IGF-II(63-72)), as well as two lysine mutants [(IGF-I(66-75)Lys(70)) and (IGF-II(63-72)Lys(67))]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I(66-75) and IGF-II(63-72), suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I(66-75)Lys(70) and IGF-II(63-72)Lys(67), were cleaved more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of pituitary adenylate-cyclase-activating polypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR downward arrow (where X denotes any other residue). As expected, p-chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu(2+)>Hg(2+)>Zn(2+) Ni(2+)>Co(2+)), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (K(i) 0.75 microM) and Arg-Ser-Lys-Arg-SC (K(i) 11.4 microM).

Amino Acid Sequence↗

PubMed2005

Dual regulation of the LDL receptor--some clarity and new questions.

Upregulation of the expression of the LDL receptor (LDLR) gene does not always lead to an increase in the LDLR protein. While the mRNA expression of the LDLR and the newly studied proprotein convertase PCSK9 are coordinately upregulated in absence of sterols, the latter proteinase apparently enhances the degradation of the LDLR protein.

Animals↗