N-glycosylation controls trafficking, zymogen activation and substrate processing of proprotein convertases PC1/3 and subtilisin kexin isozyme-1

The limited proteolysis of proteins by the proprotein convertases (PCs) is a common means of producing bioactive proteins or peptides. The PCs are associated with numerous human pathologies and their activity can be reduced through the use of specific inhibitors. Here, we demonstrate an alternative approach to inhibiting PCs by altering their N-glycosylation. Through site-directed mutagenesis, we show that the convertase PC1/3 contains two N-glycans, only one of which is critical for its prosegment cleavage. The exact structure of PC1/3 N-glycans does not significantly affect its zymogen activation within endocrine cells, but glycosylation of Asn(146) is critical. Processing of the PC1/3's substrate proopiomelanocortin (POMC) was used in a cell-based assay to screen a collection of 45 compounds structurally related to known glycosidase inhibitors. Two 5-thiomannose-containing disaccharide derivatives were discovered to block PC1/3 and POMC processing into the analgesic peptide β-endorphin. These compounds also reduced the zymogen activation of the convertase subtilisin kexin isozyme-1 (SKI-1), blocked the processing of its substrate the sterol regulatory element-binding protein SREBP-2 and altered its glycosylation. Thus, modification of PC glycosylation may also be a means of blocking their activity, an effect which, in the case of SKI-1, may be of possible therapeutic use since SREBP-2 regulates sterol levels including cholesterol biosynthesis and its metabolism.     

Multibranch and pseudopeptide approach for design of novel inhibitors of subtilisin kexin isozyme-1

Here we developed small molecule inhibitors of SKI-1/S1P enzyme of the Proprotein Convertase family following two approaches. One involves the assembly of multi-branch peptides while the other utilizes the insertion of alkyloxy pseudo peptide bond at P1-P1' cleavage position. In first approach, 2 and 4-branch peptides were designed based on the human (h) SKI-1(128-137) sequence, located N-terminal to its secondary activation site (K(137) downward arrow L). The 4-branch peptide exhibited the highest SKI-1 inhibitory property (IC(50) = 0.9 microM) with approximately 8.6 and 1.3-fold more potency than the corresponding single and 2-branch peptides, respectively. In the second strategy, an oxymethylene containing unnatural amino acid such as aminooxy-acetic acid (Aoaa) or 8-amino-3, 6 dioxa-octanoic acid (Adoa) was introduced substituting P1, P1' or both residues of hSKI-1(183-190) and hSKI-1(178-190) segments. These domains contain the same primary hSKI-1 activation site L(186) downward arrow R. Among those tested, P7-Tyr mutant [(178)GRYSSRRL(Adoa)AIP(190)] exhibited higher SKI-1 inhibitory activity (K(i)in low microM). Circular dichroism (CD) spectra of SKI-1 inhibitors showed interactions of varying degrees between the enzyme and the inhibitor consistent with the observed inhibition profile. A 3D-homology model structure of SKI-1 catalytic domain indicated a broad catalytic pocket.     

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

Aminoethyl benzenesulfonyl fluoride and its hexapeptide (Ac-VFRSLK) conjugate are both in vitro inhibitors of subtilisin kexin isozyme-1

Using a number of intramolecularly quenched fluorogenic (IQF) substrates encompassing the subtilisin kexin isozyme-1 (SKI-1)-mediated cleavage sites of various viral glycoproteins, it is revealed that 4-[2-Aminoethyl BenzeneSulfonylFluoride (AEBSF) can inhibit the proteolytic activity of SKI-1 mostly in a competitive manner. The measured IC50 values range from 200 to 800 nM depending on the nature of the substrate used. This is the first in vitro demonstration of a non-peptide inhibitor of SKI-1. In an effort to enhance the selectivity and potency of SKI-1 inhibition, a hexapeptidyl derivative containing SKI-1 consensus sequence, Ac-Val-Phe-Arg-Ser-Leu-Lys-AEBSF, was prepared. The peptide sequence was derived from the primary auto-activation site of prodomain of SKI-1 itself terminating at Leu-Lys138 and contains the crucial P4-basic and P2 alkyl side chain containing hydrophobic amino acids. Like AEBSF, the hexapeptidyl-AEBSF analog blocked SKI-1 cleavages of all IQF-substrates tested but with enhanced efficiency.     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1

We have studied the binding and interaction of the peptide E1_FP with various model membranes. E1_FP is derived from the amino acid segment 274-291 of the hepatitis C virus envelope glycoprotein E1, which was previously proposed to host the peptide responsible for fusion to target membranes. In the present study we addressed the changes which take place upon E1_FP binding in both the peptide and the phospholipid bilayer, respectively, through a series of complementary experiments. We show that peptide E1_FP binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane and interacts preferentially with cholesterol. The capability of modifying the biophysical properties of model membranes supports its role in HCV-mediated membrane fusion and suggests that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.     

Dissection of the role of the stable signal peptide of the arenavirus envelope glycoprotein in membrane fusion

The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition.     

Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.     

Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755 kDa. The NH₂-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K_cat/K_m) towards natural, modified, and synthesis substrates with optimal activity at 60 °C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg²⁺, Ca²⁺ and Ba²⁺. The structural properties of AprB, like a higher ratio of R/(R + K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN′, AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.     

Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.     

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.     

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.