Furin Inhibitor Research Articles

Furin Overexpression Suppresses Tumor Growth and Predicts a Better Postoperative Disease-Free Survival in Hepatocellular Carcinoma

Furin is a member of the pro-protein convertase family. It processes several growth regulatory proteins into their active forms, which are critical to tumor progression, metastasis, and angiogenesis. Furin over-expression could occur in liver cancer and a previous study showed that over-expression of furin promoted HepG2 cell invasion in tail vein xenograft models. However, the clinical relevance of furin expression in hepatocellular carcinoma (HCC) remained unknown. Surprisingly, in a postoperative survival analysis for HCC patients, it was found that the tumor/non-tumor (T/N) ratio of furin expression ≥ 3.5 in HCC tissues predicted a better postoperative disease-free survival (DFS) (P = 0.010; log-rank test). Furthermore, subcutaneous xenograft experiments demonstrated a significant suppression effect of tumor growth in the furin-overexpressed xenografts (Huh7-Furin) compared to the mock control. Administration of a synthetic furin inhibitor for inhibition of the pro-protein convertase activity, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decRVKR-CMK), to the Huh7-Furin xenograft bearing mice restored the repression effect of tumor growth. In contrast, administration of decRVKR-CMK to the mock Huh7 xenograft bearing mice showed no change in growth rate. In conclusion, furin overexpression inhibited HCC tumor growth in a subcutaneous xenograft model and predicted a better postoperative DFS in clinical analysis.

Highly Potent Inhibitors of Proprotein Convertase Furin as Potential Drugs for Treatment of Infectious Diseases

Optimization of our previously described peptidomimetic furin inhibitors was performed and yielded several analogs with a significantly improved activity. The most potent compounds containing an N-terminal 4- or 3-(guanidinomethyl)phenylacetyl residue inhibit furin with K(i) values of 16 and 8 pM, respectively. These analogs inhibit other proprotein convertases, such as PC1/3, PC4, PACE4, and PC5/6, with similar potency, whereas PC2, PC7, and trypsin-like serine proteases are poorly affected. Incubation of selected compounds with Madin-Darby canine kidney cells over a period of 96 h revealed that they exhibit great stability, making them suitable candidates for further studies in cell culture. Two of the most potent derivatives were used to inhibit the hemagglutinin cleavage and viral propagation of a highly pathogenic avian H7N1 influenza virus strain. The treatment with inhibitor 24 (4-(guanidinomethyl)phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide) resulted in significantly delayed virus propagation compared with an inhibitor-free control. The same analog was also effective in inhibiting Shiga toxin activation in HEp-2 cells. This antiviral effect, as well as the protective effect against a bacterial toxin, suggests that inhibitors of furin or furin-like proprotein convertases could represent promising lead structures for future drug development, in particular for the treatment of infectious diseases.

Limitations of Inhibitory Activities of Polyphenols on Furin-Mediated Substrate Processing

Recently, selected polyphenols were reported to exert proprotein convertase (PC) inhibitory activities on in vitro cleavage of a fluorogenic peptide substrate and it was concluded that this anti-protease activity might be responsible for the reported anti-cancer properties of these polyphenols. This prompted investigations to identify PC inhibiting polyphenols that could affect IGF-1R-mediated tumorigenesis since pro-IGF-1R is bioactivated by PCs like furin. Initial screening of polyphenols for their impact on in vitro cleavage of fluorogenic peptide substrate Pyr-RTKR-AMC by human furin (hfurin(573)) indeed revealed varying inhibitory effects. (-)EGCG, chrysin, and quercetin, were subsequently evaluated using uncleaved diphtheria toxin as substrate in vitro. However, none displayed any inhibitory impact on processing. Binding of (-)EGCG to both furin and the diphtheria toxin protein was demonstrated. Subsequently, it was found that for seven polyphenols tested, addition of casein or gamma globulin led to reduction or even annihilation of in vitro Pyr- RTKR-AMC cleavage inhibition. No such effect was seen with the furin inhibitor nona-D-arginine. Western blot studies to investigate possible effects of selected polyphenols on processing in cells of the tumorigenesis-linked proproteins pro-IGF-1R and pro-GPC3 also revealed no inhibitory effects. In conclusion, our results confirm the reported PC inhibitory effects of polyphenols on fluorogenic peptide substrate cleavage in vitro. However, the data show that polyphenolic inhibitory effects on hfurin(573)-mediated in vitro fluorogenic peptide substrate cleavage cannot be extrapolated to similar effects on processing of genuine proproteins, whether in vitro or in cells. This undermines the anti-protease rationale for the reported polyphenolic anti-cancer properties.

Suppression of Furin by Interferon-γ and the Impact on Hepatitis B Virus Antigen Biosynthesis in Human Hepatocytes

The roles of furin and intrahepatic cytokines in chronic heptatitis B virus (HBV) infection remain largely unknown. Here, we examined the relations between furin, IL-10, IL-12β, interferon (IFN)-γ, programed death (PD)-1, programed death ligand (PD-L)1, and the suppression of hepatitis B e antigen (HBeAg) and surface antigen (HBsAg) biosynthesis. Liver biopsies were performed on 20 chronically HBV-infected (15 HBeAg-positive and 5 HBeAg-negative) patients to assess liver inflammation/fibrosis, and mRNA levels of furin, IL-10, IL-12β, IFN-γ, PD-1, and PD-L1 were assessed by quantitative real-time PCR. IFN-γ mRNA abundance was associated with lower furin mRNA levels and higher PD-1 and PD-L1 mRNA levels in liver tissue from HBeAg-positive patients. IL-10 and IL-12β mRNA levels positively correlated with IFN-γ expression levels (P < 0.05). PD-L1 and furin mRNA levels were further assessed in IFN-γ-stimulated hepatoma cell lines with (HepG2.2.15 cells) and without (HepG2 and Huh7 cells) HBV replication. IFN-γ enhanced PD-L1 expression in hepatoma cells. In HepG2.2.15 cells, IFN-γ further suppressed furin and HBeAg expression. Furin inhibition and knockdown in HepG2.2.15 cells also down-regulated HBeAg and HBsAg biosynthesis. These data suggest that IFN-γ modulates the inflammatory response to avoid excessive hepatocyte damage through the enhancement of PD-1/PD-L1 expression, whereas furin suppression may contribute to a reduction in HBeAg/HBsAg biosynthesis.

Furin inhibitors: Importance of the positive formal charge and beyond

Furin is the prototype member of the proprotein convertases superfamily. Proprotein convertases are associated with hormonal response, neural degeneration, viral and bacterial activation, and cancer. Several studies over the last decade have examined small molecules, natural products, peptides and peptide derivatives as furin inhibitors. Currently, subnanomolar inhibition of furin is possible. Herein, we report the analysis of 115 furin inhibitors reported in the literature. Analysis of the physicochemical properties of these compounds highlights the dependence of the inhibitory potency with the total formal charge and also shows how the most potent (peptide-based) furin inhibitors have physicochemical properties similar to drugs. In addition, we report docking studies of 26 furin inhibitors using Glide XP. Inspection of binding interactions shows that the two putative binding modes derived from our study are reasonable. Analysis of the binding modes and protein–ligand interaction fingerprints, used here as postdocking procedure, shows that electrostatic interactions predominate on S1, S2 and S4 subsites but are seldom in S3. Our models also show that the benzimidamide group, present in the most active inhibitors, can be accommodated in the S1 subsite. These results are valuable for the design of new furin inhibitors.

Abstract 4627: Generation and characterization of non-competitive furin-inhibiting nanobodies

Abstract Furin belongs to a family of seven closely related subtilisin-like serine endoproteases, known as proprotein convertases (PCs), PC1/3, PC2, PC4, PC5/6, PACE4 and PC7. The physiological role of furin is to cleave and hence activate a large variety of proproteins. Therefore, it is not surprising that it plays a major role in many pathologies such as cancer and infectious diseases. Furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although thus far none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy chain antibodies against catalytically active furin were developed as specific furin inhibitors. Nanobodies derived from these antibodies bind only to mouse and human furin but not to other PCs. In addition, upon overexpression in cell lines, these nanobodies can inhibit the cleavage of three different furin substrates, TGFβ, BAFF and GPC3. The purified nanobodies can also inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but do not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode of action based on steric hindrance. The inhibitory constant of the purified nanobodies was shown to be in the micromolar range and the nanobodies are non-competitive inhibitors as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies can protect HEK293T cells from diphtheria toxin-induced cytotoxicity as efficiently as the well characterized PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-chain nanobodies represent the first generation of highly specific, non-competitive furin inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4627. doi:1538-7445.AM2012-4627

Post-translational regulated and hypoxia-responsible VEGF plasmid for efficient secretion

Gene therapy using angiogenic genes has emerged as a potentially viable alternative treatment strategy for myocardial ischemia. Non-specific expression of angiogenic genes, however, may result in side effects, including the growth of occult tumors. Regulation of gene expression may help to avoid the occurrence of these side effects. In this study, a plasmid expressing vascular endothelial growth factor (VEGF) was constructed with an oxygen dependent degradation (ODD) domain and a secretion signal peptide (SP) in order to stabilize the VEGF protein and facilitate the secretion of VEGF protein, specifically under hypoxic conditions. We found that this plasmid, pβ-SP-ODD-VEGF, expresses the SP-ODD-VEGF protein at increased levels under hypoxic conditions compared to normoxic conditions. Since the size of the ODD domain is almost the same as that of VEGF, the ODD-VEGF fusion protein may have lower secretion efficiency. To address this issue, a furin recognition site was located between the ODD domain and the VEGF site to facilitate elimination of the SP-ODD domain from the fusion protein before its secretion. This optimizes the likelihood that the VEGF secreted from the target cells will be wild-type VEGF. Treatment with a furin inhibitor reduced the secretion efficiency of the VEGF, indicating that furin digestion increases the secretion of VEGF. The secreted wild-type VEGF facilitated the growth of endothelial cells more efficiently under hypoxic conditions than normoxic conditions. These results suggest that this plasmid, pβ-SP-ODD-VEGF, warrants further study as a more efficient form of hypoxia-inducible gene therapy for the treatment of myocardial ischemia.

Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

Exploiting pleiotropic activities of semaphorins as multi‐target therapies for cancer

Exploiting pleiotropic activities of semaphorins as multi‐target therapies for cancer

Mutation of the F-Protein Cleavage Site of Avian Paramyxovirus Type 7 Results in Furin Cleavage, Fusion Promotion, and Increased Replication In Vitro but Not Increased Replication, Tissue Tropism, or Virulence in Chickens

We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.

Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)–Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell–matrix interface.

A novel peptide to enhance recombinant BMP-2 production in mammalian cell cultures

Materials and methods Two stable cell lines expressing the hBMP2 gene, CHOBMP2 and HEK-BMP2, were cultured in the presence of IND-1 in short-term (24 h, multi-well) and long-term (two-month, perfusion flasks) cultures. The rhBMP-2 produced was characterized by Western blot and its activity assessed using the C2C12 cell-based assay. The amount of proBMP-2 and mature BMP-2 produced was quantified by ELISA. The mRNA level of BMP-2 and furin in cells treated with or without IND-1 was compared by real-time RT-PCR. Cellular uptake of IND-1 was estimated by measuring the fluorescence of cell lysates following incubation with FITC labeled IND-1. Cellular PC activity post IND-1 incubation was measured using the Boc-RVRR-AMC substrate. Furin-specific siRNA was used to knock down the furin expression in CHO-BMP2 cells and its effect on the rhBMP-2 production was determined. Results Stably transfected CHO-BMP2 cells secreted 36 kDa rhBMP-2 dimers that were biologically active. In 24 h cell cultures, IND-1 treated cells produced significantly greater amounts of proBMP-2 (≥ 10-fold, P <0.001 )a nd mature BMP-2 (≥ 3-fold, P <0.001) in their conditioned medium (Figure 1). In long-term CHO-BMP2 culture, IND-1 continued to increase the yields of BMP-2 (≥ 50%) and proBMP-2 (≥ 2-fold) without affecting cell growth or viability. IND-1 treatment had no effect on the mRNA level of BMP-2 and furin, indicating IND-1 affects rhBMP-2 yield post-transcriptionally. While IND-1 was taken up by the cells and inhibited PC activity when added directly to the cell lysates, cells cultured with IND-1 showed no changes in their PC activity at doses 50 times higher than required to affect BMP-2 yields. Furthermore, knockdown of furin at both the mRNA (≥ 80%, P <0.001) and the protein level (≥ 70%, P <0.001), did not affect rhBMP-2 yields. These results suggest that furin inhibition is most l ikely not the mechanism by which IND-1 enhances rhBMP-2 yields. Conclusions The addition of a novel peptide IND-1 to the cell culture medium significantly enhanced the yields of both pro- and mature BMP-2 in stably transfected CHO and HEK cell lines. These increases were sustainable over an extended time period with regular IND-1 treatments. However, the enhanced rhBMP-2 yield is unlikely due to the well-established role of polyarginines as furin inhibitors.

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

BackgroundChikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.ResultsExpression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.ConclusionsChikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

New substrate analogue furin inhibitors derived from 4-amidinobenzylamide

A series of new peptidomimetic furin inhibitors was synthesized, which was derived from our previously described lead structure phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (1). Substitution of Val by other amino acid residues revealed several highly potent furin inhibitors with K(i) values of less than 2nM, containing guanidinoalanine, Ile, Phe or Tyr in the P3 position. The replacement of the P2 Arg by Lys was also well accepted, whereas the incorporation of D-amino acids at various positions resulted in poor inhibitors. The use of the 4-amidinobenzylamide group provides convenient synthetic access to stable proprotein convertase inhibitors and derivatives as biochemical tools and for further studies in cell culture.

Processing of Proaugurin Is Required to Suppress Proliferation of Tumor Cell Lines

Augurin is a secretory molecule produced in pituitary, thyroid, and esophagus and implicated in a wide array of physiological processes, from ACTH release to tumor suppression. However, the specific proaugurin-derived peptides present in various cell types are not yet known. In order to shed light on the posttranslational modifications required for biological activity, we here describe the posttranslational processing of proaugurin in AtT-20 and Lovo cells and identify proaugurin-derived products generated by convertases. In vitro cleavage of proaugurin with proprotein convertases produced multiple peptides, including a major product with a mass of 9.7 kDa by mass spectrometry. Metabolic labeling of C-terminally tagged proaugurin in AtT-20 and AtT-20/PC2 cells resulted in a major 15-kDa tagged form on SDS-PAGE, which likely corresponds to the 9.7-kDa in vitro fragment, with the added tag, its linker, and posttranslational modification(s). The secretion of neither proaugurin nor this cleavage product was stimulated by forskolin, indicating its lack of storage in regulated secretory granules and lack of cleavage by PC2. Incubation of cells with the furin inhibitor nona-d-arginine resulted in impaired cleavage of proaugurin, whereas metalloprotease inhibitors did not affect proaugurin proteolysis. These data support the idea that proaugurin is cleaved by furin and secreted via the constitutive secretory pathway. Interestingly, proaugurin was sulfated during trafficking; sulfation was completely inhibited by brefeldin A. Proliferation assays with three different tumor cell lines demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to suppress cell proliferation.

Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase

Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.

New furin inhibitors based on weakly basic amidinohydrazones

A novel series of amidinohydrazone-derived furin inhibitors was prepared; the most potent compounds 17 and 21 inhibit furin with K(i) values of 0.46 and 0.59μM, respectively. In contrast to inhibitor 17, which still contains a guanidino residue, compound 21 possesses only weakly basic amidinohydrazone groups.

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.

Blockade of Furin Activity and Furin-Induced Tumor Cells Malignant Phenotypes By The Chemically Synthesized Human Furin Prodomain

Processing of cancer-associated precursor proteins such as growth factors by the Proprotein Convertase furin is an important cellular process for acquisition of malignant phenotype and metastatic potential of tumor cells. Furin is inhibited by its own prodomain protein which also plays role in the folding of mature furin. Herein, the complete 83-mer furin prodomain protein was chemically synthesized for the first time by solid phase peptide chemistry and its effects on furin activity and tumor cells malignant phenotypes were evaluated. It inhibited furin activity in a competitive manner with low nanomolar inhibition constant (Ki). Expression of furin prodomain cDNA in tumor cells or cells incubated with the corresponding protein blocked furin-cleavage of proPDGF-A. This was associated with significant reduction in tumor cells proliferation, migration as well as invasion. Interestingly shorter synthetic furin prodomain peptides derived from either its primary or secondary activation site alone weakly inhibited furin and displayed limited effects on tumor cells. This suggests that the combined presence of the above two prodomain segments is crucial for prodomain's potent furin-inhibitory and hence anticancer activities.