Journal articles on the topic 'Furin site'
To see the other types of publications on this topic, follow the link: Furin site.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Furin site.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Tian, Sun. "A 20 Residues Motif Delineates the Furin Cleavage Site and its Physical Properties May Influence Viral Fusion." Biochemistry Insights 2 (January 2009): BCI.S2049. http://dx.doi.org/10.4137/bci.s2049.
Full textAbstract:
Furin is a proprotein convertase that proteolytically cleaves protein precursors to yield functional proteins. Efficient cleavage depends on the presence of a specific sequence motif on the substrate. Currently, the cleavage site motif is described as a four amino acid pattern: R-X-[K/R]-R⇓. However, not all furin cleavage recognition sites can be described by this pattern and not all R-X-[K/R]-R⇓ sites are cleaved by furin. Since many furin substrates are involved in the pathogenesis of viral infection and human diseases, it is important to accurately characterize the furin cleavage site motif. In this study, the furin cleavage site motif was characterized using statistical analysis. The data were interpreted within the 3D crystal structure of the furin catalytic domain. The results indicate that the furin cleavage site motif is comprised of about 20 residues, P14-P6′. Specific physical properties such as volume, charge, and hydrophilicity are required at specific positions. The furin cleavage site motif is divided into two parts: 1) one core region (8 amino acids, positions P6-P2′) packed inside the furin binding pocket; 2) two polar regions (8 amino acids, positions P7–P14; and 4 amino acids, positions P3′-P6′) located outside the furin binding pocket. The physical properties of the core region contribute to the binding strength of the furin substrate, while the polar regions provide a solvent accessible environment and facilitate the accessibility of the core region to the furin binding pocket. This furin cleavage site motif also revealed a dynamic relationship linking the evolution of physical properties in region P1′-P6′ of viral fusion peptides, furin cleavage efficacy, and viral infectivity.
2
Yamada, Yoshiyuki, and Ding Xiang Liu. "Proteolytic Activation of the Spike Protein at a Novel RRRR/S Motif Is Implicated in Furin-Dependent Entry, Syncytium Formation, and Infectivity of Coronavirus Infectious Bronchitis Virus in Cultured Cells." Journal of Virology 83, no. 17 (June 24, 2009): 8744–58. http://dx.doi.org/10.1128/jvi.00613-09.
Full textAbstract:
ABSTRACT The spike (S) protein of the coronavirus (CoV) infectious bronchitis virus (IBV) is cleaved into S1 and S2 subunits at the furin consensus motif RRFRR537/S in virus-infected cells. In this study, we observe that the S2 subunit of the IBV Beaudette strain is additionally cleaved at the second furin site (RRRR690/S) in cells expressing S constructs and in virus-infected cells. Detailed time course experiments showed that a peptide furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, blocked both viral entry and syncytium formation. Site-directed mutagenesis studies revealed that the S1/S2 cleavage by furin was not necessary for, but could promote, syncytium formation by and infectivity of IBV in Vero cells. In contrast, the second site is involved in the furin dependence of viral entry and syncytium formation. Mutations of the second site from furin-cleavable RRRR/S to non-furin-cleavable PRRRS and AAARS, respectively, abrogated the furin dependence of IBV entry. Instead, a yet-to-be-identified serine protease(s) was involved, as revealed by protease inhibitor studies. Furthermore, sequence analysis of CoV S proteins by multiple alignments showed conservation of an XXXR/S motif, cleavable by either furin or other trypsin-like proteases, at a position equivalent to the second IBV furin site. Taken together, these results suggest that proteolysis at a novel XXXR/S motif in the S2 subunit might be a common mechanism for the entry of CoV into cells.
3
Cheng, Jinlong, Ye Zhao, Gang Xu, Keran Zhang, Wenfeng Jia, Yali Sun, Jing Zhao, Jia Xue, Yanxin Hu, and Guozhong Zhang. "The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism." Viruses 11, no. 10 (October 22, 2019): 972. http://dx.doi.org/10.3390/v11100972.
Full textAbstract:
Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2′ site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2′ site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2′ site (furin-S2′ site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2′ site as a determinant of neurotropism.
4
Siner, Joshua I., Julie M. Crudele, Courtney T. Connolly, Shangzhen Zhou, Elizabeth P. Merricks, Timothy C. Nichols, Rodney M. Camire, and Valder R. Arruda. "Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy." Blood 124, no. 21 (December 6, 2014): 105. http://dx.doi.org/10.1182/blood.v124.21.105.105.
Full textAbstract:
Abstract The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion. We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD. To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin. Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025). These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs. In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. Disclosures No relevant conflicts of interest to declare.
5
Papa, Guido, Donna L. Mallery, Anna Albecka, Lawrence G. Welch, Jérôme Cattin-Ortolá, Jakub Luptak, David Paul, et al. "Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion." PLOS Pathogens 17, no. 1 (January 25, 2021): e1009246. http://dx.doi.org/10.1371/journal.ppat.1009246.
Full textAbstract:
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.
6
Wanyiri, Jane W., Roberta O'Connor, Geneve Allison, Kami Kim, Anne Kane, Jiazhou Qiu, Andrew G. Plaut, and Honorine D. Ward. "Proteolytic Processing of the Cryptosporidium Glycoprotein gp40/15 by Human Furin and by a Parasite-Derived Furin-Like Protease Activity." Infection and Immunity 75, no. 1 (October 16, 2006): 184–92. http://dx.doi.org/10.1128/iai.00944-06.
Full textAbstract:
ABSTRACT The apicomplexan parasite Cryptosporidium causes diarrheal disease worldwide. Proteolytic processing of proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we described gp40/15, a Cryptosporidium sp. glycoprotein that is proteolytically cleaved to yield two surface glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study, we showed that biosynthetically labeled gp40/15 is processed in Cryptosporidium parvum-infected HCT-8 cells. We identified a putative furin cleavage site RSRR↓ in the deduced amino acid sequence of gp40/15 from C. parvum and from all Cryptosporidium hominis subtypes except subtype 1e. Both human furin and a protease activity present in a C. parvum lysate cleaved recombinant C. parvum gp40/15 protein into 2 peptides, identified as gp40 and gp15 by size and by immunoreactivity with specific antibodies. C. hominis gp40/15 subtype 1e, in which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR↓) and was also cleaved by both furin and the C. parvum lysate. Site-directed mutagenesis of the C. parvum RSRR sequence to ASRR resulted in inhibition of cleavage by furin and the C. parvum lysate. Cleavage of recombinant gp40/15 and a synthetic furin substrate by the C. parvum lysate was inhibited by serine protease inhibitors, by the specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators, suggesting that the parasite expresses a Ca2+ dependent, furin-like protease activity. The furin inhibitor Dec-RVKR-cmk decreased C. parvum infection of HCT-8 cells, suggesting that a furin-like protease activity may be involved in mediating host-parasite interactions.
7
Geiselhart, Verena, Patrizia Bastone, Tore Kempf, Martina Schnölzer, and Martin Löchelt. "Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein." Journal of Virology 78, no. 24 (December 15, 2004): 13573–81. http://dx.doi.org/10.1128/jvi.78.24.13573-13581.2004.
Full textAbstract:
ABSTRACT The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.
8
Cui, Yanzhen, Renee Hackenmiller, Linnea Berg, François Jean, Takuya Nakayama, Gary Thomas, and Jan L. Christian. "The activity and signaling range of mature BMP-4 is regulated by sequential cleavage at two sites within the prodomain of the precursor." Genes & Development 15, no. 21 (November 1, 2001): 2797–802. http://dx.doi.org/10.1101/gad.940001.
Full textAbstract:
Proteolytic maturation of proBMP-4 is required to generate an active signaling molecule. We show that proBMP-4 is cleaved by furin in a sequential manner. Cleavage at a consensus furin site adjacent to the mature ligand domain allows for subsequent cleavage at an upstream nonconsensus furin site within the prodomain. BMP-4 synthesized from precursor in which the upstream site is noncleavable is less active, signals at a shorter range, and accumulates at lower levels than does BMP-4 cleaved from native precursor. Conversely, BMP-4 cleaved from precursor in which both sites are rapidly cleaved is more active and signals over a greater range. Differential use of the upstream cleavage site could provide for tissue-specific regulation of BMP-4 activity and signaling range.
9
Gendron, Fernand-Pierre, Sébastien Mongrain, Patrick Laprise, Stéphanie McMahon, Claire M. Dubois, Mylène Blais, Claude Asselin, and Nathalie Rivard. "The CDX2 transcription factor regulates furin expression during intestinal epithelial cell differentiation." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 2 (February 2006): G310—G318. http://dx.doi.org/10.1152/ajpgi.00217.2005.
Full textAbstract:
CDX2, a member of the caudal family of transcription factors, is involved in enterocyte lineage specification. CDX2 activates many intestine-specific genes, such as sucrase-isomaltase and lactase-phlorizin hydrolase (LPH), and adhesion proteins, namely, LI-cadherin and claudin-2. In this study, we show that the proprotein convertase furin, involved in proteolytic maturation of proprotein substrates including LPH and cell surface proteins, is a CDX2 target. Indeed, expression of the rat furin homolog was induced 1.5-fold, as determined by microarray experiments that compared control with CDX2-expressing intestinal epithelial cells (IEC-6). As determined by transient transfection assays in Caco-2/15 cells, the furin P1 promoter 1.3-kb fragment between SacI and NheI was essential for CDX2 transcriptional activation. Electrophoretic mobility shift/supershift assays followed by site-specific mutagenesis and chromatin immunoprecipitation identified the CDX DNA-binding site (CBS)2 sequence from nt −1827 to −1821 as the major CBS involved in furin P1 promoter activation. Increased furin mRNA and protein expression correlated with both CDX2 expression and intestinal epithelial cell differentiation. In addition, furin mRNAs were detected predominantly in differentiated epithelial cells of the villus, as determined by in situ hybridization. Treatment of Caco-2/15 cells with a furin inhibitor led to inhibition of LPH activity. Morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush-border formation were strongly attenuated by furin inhibition. These results suggest that CDX2 regulates furin expression in intestinal epithelial cells. Furin may be important in modulating the maturation and/or activation of key factors involved in enterocyte differentiation.
10
Mjokane, Nozethu, Maphori Maliehe, Olufemi S. Folorunso, Adepemi O. Ogundeji, Onele M. N. Gcilitshana, Jacobus Albertyn, Carolina H. Pohl, and Olihile M. Sebolai. "Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein." Cells 11, no. 3 (January 27, 2022): 437. http://dx.doi.org/10.3390/cells11030437.
Full textAbstract:
In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.
11
Thomas, Gary, Frédéric Couture, and Anna Kwiatkowska. "The Path to Therapeutic Furin Inhibitors: From Yeast Pheromones to SARS-CoV-2." International Journal of Molecular Sciences 23, no. 7 (March 22, 2022): 3435. http://dx.doi.org/10.3390/ijms23073435.
Full textAbstract:
The spurious acquisition and optimization of a furin cleavage site in the SARS-CoV-2 spike protein is associated with increased viral transmission and disease, and has generated intense interest in the development and application of therapeutic furin inhibitors to thwart the COVID-19 pandemic. This review summarizes the seminal studies that informed current efforts to inhibit furin. These include the convergent efforts of endocrinologists, virologists, and yeast geneticists that, together, culminated in the discovery of furin. We describe the pioneering biochemical studies which led to the first furin inhibitors that were able to block the disease pathways which are broadly critical for pathogen virulence, tumor invasiveness, and atherosclerosis. We then summarize how these studies subsequently informed current strategies leading to the development of small-molecule furin inhibitors as potential therapies to combat SARS-CoV-2 and other diseases that rely on furin for their pathogenicity and progression.
12
Galanopoulou, A. S., N. G. Seidah, and Y. C. Patel. "Direct role of furin in mammalian prosomatostatin processing." Biochemical Journal 309, no. 1 (July 1, 1995): 33–40. http://dx.doi.org/10.1042/bj3090033.
Full textAbstract:
We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.
13
Zhao, Ming, Lyn Gold, Ann M. Ginsberg, Li-Fang Liang, and Jurrien Dean. "Conserved Furin Cleavage Site Not Essential for Secretion and Integration of ZP3 into the Extracellular Egg Coat of Transgenic Mice." Molecular and Cellular Biology 22, no. 9 (May 1, 2002): 3111–20. http://dx.doi.org/10.1128/mcb.22.9.3111-3120.2002.
Full textAbstract:
ABSTRACT The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR → ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.
14
Pearce, Kenneth H., Laurie K. Overton, Robert T. Gampe, George B. Barrett, J. David Taylor, David D. McKee, Nino Campobasso, Robert T. Nolte, and Robert A. Reid. "BacMam production and crystal structure of nonglycosylated apo human furin at 1.89 Å resolution." Acta Crystallographica Section F Structural Biology Communications 75, no. 4 (March 13, 2019): 239–45. http://dx.doi.org/10.1107/s2053230x19001419.
Full textAbstract:
Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
15
Passero, Christopher J., Gunhild M. Mueller, Michael M. Myerburg, Marcelo D. Carattino, Rebecca P. Hughey, and Thomas R. Kleyman. "TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site." American Journal of Physiology-Renal Physiology 302, no. 1 (January 1, 2012): F1—F8. http://dx.doi.org/10.1152/ajprenal.00330.2011.
Full textAbstract:
The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.
16
Hossain, Md Shahadat, Mahafujul Islam Quadery Tonmoy, Atqiya Fariha, Md Sajedul Islam, Arpita Singha Roy, Md Nur Islam, Kumkum Kar, Mohammad Rahanur Alam, and Md Mizanur Rahaman. "Prediction of the Effects of Variants and Differential Expression of Key Host Genes ACE2, TMPRSS2, and FURIN in SARS-CoV-2 Pathogenesis: An In Silico Approach." Bioinformatics and Biology Insights 15 (January 2021): 117793222110546. http://dx.doi.org/10.1177/11779322211054684.
Full textAbstract:
A new strain of the beta coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is solely responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Although several studies suggest that the spike protein of this virus interacts with the cell surface receptor, angiotensin-converting enzyme 2 (ACE2), and is subsequently cleaved by TMPRSS2 and FURIN to enter into the host cell, conclusive insight about the interaction pattern of the variants of these proteins is still lacking. Thus, in this study, we analyzed the functional conjugation among the spike protein, ACE2, TMPRSS2, and FURIN in viral pathogenesis as well as the effects of the mutations of the proteins through the implementation of several bioinformatics approaches. Analysis of the intermolecular interactions revealed that T27A (ACE2), G476S (receptor-binding domain [RBD] of the spike protein), C297T (TMPRSS2), and P812S (cleavage site for TMPRSS2) coding variants may render resistance in viral infection, whereas Q493L (RBD), S477I (RBD), P681R (cleavage site for FURIN), and P683W (cleavage site for FURIN) may lead to increase viral infection. Genotype-specific expression analysis predicted several genetic variants of ACE2 (rs2158082, rs2106806, rs4830971, and rs4830972), TMPRSS2 (rs458213, rs468444, rs4290734, and rs6517666), and FURIN (rs78164913 and rs79742014) that significantly alter their normal expression which might affect the viral spread. Furthermore, we also found that ACE2, TMPRSS2, and FURIN proteins are functionally co-related with each other, and several genes are highly co-expressed with them, which might be involved in viral pathogenesis. This study will thus help in future genomics and proteomics studies of SARS-CoV-2 and will provide an opportunity to understand the underlying molecular mechanism during SARS-CoV-2 pathogenesis.
17
Semenov, Alexander G., Natalia N. Tamm, Karina R. Seferian, Alexander B. Postnikov, Natalia S. Karpova, Daria V. Serebryanaya, Ekaterina V. Koshkina, Mihail I. Krasnoselsky, and Alexey G. Katrukha. "Processing of Pro–B-Type Natriuretic Peptide: Furin and Corin as Candidate Convertases2." Clinical Chemistry 56, no. 7 (July 1, 2010): 1166–76. http://dx.doi.org/10.1373/clinchem.2010.143883.
Full textAbstract:
Abstract Background: B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. Methods: We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. Results: Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1–32, whereas corin-mediated cleavage led to the production of BNP 4–32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. Conclusions: Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.
18
Ashworth, J. L., V. Kelly, M. J. Rock, C. A. Shuttleworth, and C. M. Kielty. "Regulation of fibrillin carboxy-terminal furin processing by N-glycosylation, and association of amino- and carboxy-terminal sequences." Journal of Cell Science 112, no. 22 (November 15, 1999): 4163–71. http://dx.doi.org/10.1242/jcs.112.22.4163.
Full textAbstract:
The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12–15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.
19
WILLNOW, Thomas E., Joan M. MOEHRING, Noel M. INOCENCIO, Thomas J. MOEHRING, and Joachim HERZ. "The low-density-lipoprotein receptor-related protein (LRP) is processed by furin in vivo and in vitro." Biochemical Journal 313, no. 1 (January 1, 1996): 71–76. http://dx.doi.org/10.1042/bj3130071.
Full textAbstract:
The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, protease-inhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisin-type protease, to be necessary for efficient processing of LRP in cells. Furin-deficient RPE.40 cells exhibited an impaired processing of endogenous LRP and of a recombinant soluble form of the receptor containing the processing site. The processing defect in RPE.40 cells could be complemented by expression of furin from a transfected cDNA in cultured cells and by purified furin in vitro. The impaired maturation of LRP in RPE.40 cells did not affect its intracellular transport, and correlated with a slight but consistent reduction in the endocytosis of LRP-specific ligands. These data suggest that proteolytic processing of LRP by furin is not necessary for intracellular trafficking but might be required for normal receptor activity.
20
Dahms, Sven O., Marcelino Arciniega, Torsten Steinmetzer, Robert Huber, and Manuel E. Than. "Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism." Proceedings of the National Academy of Sciences 113, no. 40 (September 19, 2016): 11196–201. http://dx.doi.org/10.1073/pnas.1613630113.
Full textAbstract:
Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8–2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the “off state,” incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active “on state” seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin.
21
Binley, James M., Rogier W. Sanders, Aditi Master, Charmagne S. Cayanan, Cheryl L. Wiley, Linnea Schiffner, Bruce Travis, et al. "Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins." Journal of Virology 76, no. 6 (March 15, 2002): 2606–16. http://dx.doi.org/10.1128/jvi.76.6.2606-2616.2002.
Full textAbstract:
ABSTRACT In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.
22
LISSITZKY, Jean-Claude, José LUIS, Jon Scott MUNZER, Suzanne BENJANNET, Francis PARAT, Michel CHRÉTIEN, Jacques MARVALDI, and Nabil Georges SEIDAH. "Endoproteolytic processing of integrin pro-α subunits involves the redundant function of furin and proprotein convertase (PC) 5A, but not paired basic amino acid converting enzyme (PACE) 4, PC5B or PC7." Biochemical Journal 346, no. 1 (February 8, 2000): 133–38. http://dx.doi.org/10.1042/bj3460133.
Full textAbstract:
Several integrin α subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-α5, α6 and αv by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-α subunit substrates in vitro; the defective pro-α chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-α subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin α-subunit precursors. In conclusion, these results indicate that integrin α-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.
23
Zorgati, Habiba, Mårten Larsson, Weitong Ren, Adelene Y. L. Sim, Jan Gettemans, Jonathan M. Grimes, Wenfei Li, and Robert C. Robinson. "The role of gelsolin domain 3 in familial amyloidosis (Finnish type)." Proceedings of the National Academy of Sciences 116, no. 28 (June 26, 2019): 13958–63. http://dx.doi.org/10.1073/pnas.1902189116.
Full textAbstract:
In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.
24
BERGERON, Eric, Ajoy BASAK, Etienne DECROLY, and Nabil G. SEIDAH. "Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage." Biochemical Journal 373, no. 2 (July 15, 2003): 475–84. http://dx.doi.org/10.1042/bj20021630.
Full textAbstract:
The proprotein convertases (PCs) participate in the limited proteolysis of integrin α4 subunit at the H592VISKR597 ↓ ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and ↓ indicates the cleavage site), since this cleavage is inhibited by the serpin α1-PDX (α1-antitrypsin Portland). Co-expression of α4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-α4 convertases. In agreement, processing of endogenous pro-α4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-α4 convertase. Mutational analysis showed that replacement of P1 Arg597 with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type α4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-α4 and its H592R and H592K mutants in the presence or absence of the weak base, NH4Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-α4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg↓, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.
25
Bonod-Bidaud, Christelle, Mickaël Beraud, Elisabeth Vaganay, Frédéric Delacoux, Bernard Font, David J. S. Hulmes, and Florence Ruggiero. "Enzymatic cleavage specificity of the proα1(V) chain processing analysed by site-directed mutagenesis." Biochemical Journal 405, no. 2 (June 27, 2007): 299–306. http://dx.doi.org/10.1042/bj20070051.
Full textAbstract:
The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proα1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proα1(V) chain, referred to as Nα1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nα1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nα1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nα1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2′ is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.
26
Raghunath, M., E. A. Putnam, T. Ritty, D. Hamstra, E. S. Park, M. Tschodrich-Rotter, R. Peters, A. Rehemtulla, and D. M. Milewicz. "Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix." Journal of Cell Science 112, no. 7 (April 1, 1999): 1093–100. http://dx.doi.org/10.1242/jcs.112.7.1093.
Full textAbstract:
Fibrillin-1, the main component of 10–12 nm microfibrils of the extracellular matrix, is synthesized as profibrillin and proteolytically processed to fibrillin. The putative cleavage site has been mapped to the carboxy-terminal domain of profibrillin-1, between amino acids arginine 2731 and serine 2732, by a spontaneous mutation in this recognition site that prevents profibrillin conversion. This site contains a basic amino acid recognition sequence (R-G-R-K-R-R) for proprotein convertases of the furin/PACE family. In this study, we use a mini-profibrillin protein to confirm the cleavage in the carboxy-terminal domain by both fibroblasts and recombinantly expressed furin/PACE, PACE4, PC1/3 and PC2. Site-directed mutagenesis of amino acids in the consensus recognition motif prevented conversion, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Using a PACE/furin inhibitor, we show that wild-type profibrillin is not incorporated into the extracellular matrix until it is converted to fibrillin. Therefore, profibrillin-1 is the first extracellular matrix protein to be shown to be a substrate for subtilisin-like proteases, and the conversion of profibrillin to fibrillin controls microfibrillogenesis through exclusion of uncleaved profibrillin.
27
Solovyeva, N. I., T. A. Gureeva, O. S. Timoshenko, T. A. Moskvitina, and E. V. Kugaevskaya. "Furin as proprotein convertase and its role in normal and pathological biological processes." Biomeditsinskaya Khimiya 62, no. 6 (2016): 609–21. http://dx.doi.org/10.18097/pbmc20166206609.
Full textAbstract:
Furin belongs to serine intracellular Ca2+-dependent endopeptidases of the subtilisin family, also known as proprotein convertase (PC). Human furin is synthesized as zymogen with a molecular weight of 104 kDa, which is then activated by autocatalytic in two stages. This process can occur when zymogen migrates from the endoplasmic reticulum to the Golgi apparatus, where a large part of furin is accumulated. The molecular weigh t of the active furin is 98 kDa. Furin relates to enzymes with a narrow substrate specificity: it hydrolyzes peptide bonds at the site of paired basic amino acids and furin activity exhibits in a wide pH range 5-8. Its main biological function is activation of the functionally important protein precursors. It is accompanied by the launch of a cascade of reactions, which lead to appearance of biologically active molecules involved in realization of specific biological functions both in normal and in some patologicheskih processes. Furin substrates are biologically important proteins such as enzymes, hormones, growth factors and differentiation, receptors, adhesion proteins, proteins of blood plasma. Furin plays an important role in the development of processes such as proliferation, invasion, cell migration, survival, maintenance of homeostasis, embryogenesis, as well as the development of a number of pathologies, including cardiovascular, oncologic and neurodegenerative diseases. Furin and furin-like proprotein convertases participate as key factors in the realization of the regulatory functions of proteolytic enzymes, the value of which is currently being evaluated as most important in comparison with the degradative function of proteases.
28
Guimont, Philippe, Francine Grondin, and Claire M. Dubois. "Sox9-dependent transcriptional regulation of the proprotein convertase furin." American Journal of Physiology-Cell Physiology 293, no. 1 (July 2007): C172—C183. http://dx.doi.org/10.1152/ajpcell.00349.2006.
Full textAbstract:
The proprotein convertase furin participates in the maturation/bioactivation of a variety of proproteins involved in chondrogenesis events. These include parathyroid hormone-related peptide (PTHrP), an autocrine/paracrine factor that is crucial to both normal cartilage development and cartilage-related pathological processes. Despite the known importance of furin activity in the bioactivation of the polypeptides, the mechanisms that control furin regulation in chondrogenesis remain unknown. To gain insight into the molecular regulation of furin, we used the mouse prechondrogenic ATDC5 cell line, an established in vitro model of cartilage differentiation. Peak expression of both furin mRNA and furin PTHrP maturation was observed during chondrocyte nodule formation stage, an event that correlated with increased mRNA levels of Sox9, a potent high-mobility-group (HMG) box-containing transcription factor required for cartilage formation. Inhibition of furin activity led to a diminution in maturation of PTHrP, suggesting a relationship between Sox9-induced regulation of furin and chondrogenesis events. Transient transfection of Sox9 in nonchondrogenic cells resulted in a marked increase in furin mRNA and in the transactivation of the furin P1A promoter. Direct Sox9 action on the P1A promoter was narrowed down to a critical paired site with Sox9 binding capability in vitro and in vivo. Sox9 transactivation effect was inhibited by L-Sox5 and Sox-6, two Sox9 homologs also expressed in ATDC5 cells. Sox6 inhibitory effect was reduced when using Sox6-HMG-box mutants, indicating a repressive effect through direct HMG-box/DNA binding. Our work suggests a mechanism by which furin is regulated during chondrogenesis. It also adds to the complexity of Sox molecule interaction during gene regulation.
29
Xiang, Yan, and Bernard Moss. "Molluscum Contagiosum Virus Interleukin-18 (IL-18) Binding Protein Is Secreted as a Full-Length Form That Binds Cell Surface Glycosaminoglycans through the C-Terminal Tail and a Furin-Cleaved Form with Only the IL-18 Binding Domain." Journal of Virology 77, no. 4 (February 15, 2003): 2623–30. http://dx.doi.org/10.1128/jvi.77.4.2623-2630.2003.
Full textAbstract:
ABSTRACT Some poxviruses and their mammalian hosts encode homologous proteins that bind interleukin-18 (IL-18) with high affinity and inhibit IL-18-mediated immune responses. MC54L, the IL-18 binding protein of the human poxvirus that causes molluscum contagiosum, is unique in having a C-terminal tail of nearly 100 amino acids that is dispensable for IL-18 binding. When recombinant MC54L was expressed and purified via a C-terminal six-histidine tag, a shorter fragment was detected in addition to the full-length protein. This C-terminal fragment resulted from the cleavage of MC54L by cellular furin, as it was greatly diminished when furin was specifically inhibited or when a furin-deficient cell line was used for expression. Furthermore, the N- and C-terminal fragments of MC54L were generated by cleavage of the recombinant protein with furin in vitro. The furin cleavage site was mapped within a 32-amino-acid segment that is C terminal to the IL-18 binding domain. Full-length MC54L, but not the N-terminal IL-18 binding fragment, bound to cells and to purified heparin and other glycosaminoglycans that are commonly found on the cell surface and in the extracellular matrix. MC54L bound to heparin with a nanomolar Kd and could simultaneously bind to IL-18. Their different glycosaminoglycan and cell binding properties may allow the long and short forms of MC54L to inactivate IL-18 near the site of infection and at more distal locations, respectively.
30
Komoto, Satoshi, Mitsutaka Wakuda, Tomihiko Ide, Gen Niimi, Yoshimasa Maeno, Kyoko Higo-Moriguchi, and Koki Taniguchi. "Modification of the trypsin cleavage site of rotavirus VP4 to a furin-sensitive form does not enhance replication efficiency." Journal of General Virology 92, no. 12 (December 1, 2011): 2914–21. http://dx.doi.org/10.1099/vir.0.033886-0.
Full textAbstract:
The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestines in vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.
31
Spencer, J. D., N. C. J. Gibbons, M. Böhm, and K. U. Schallreuter. "The Ca2+-Binding Capacity of Epidermal Furin Is Disrupted by H2O2-Mediated Oxidation in Vitiligo." Endocrinology 149, no. 4 (January 3, 2008): 1638–45. http://dx.doi.org/10.1210/en.2007-1317.
Full textAbstract:
The Ca2+-dependent precursor convertase furin is abundantly expressed in epidermal keratinocytes and melanocytes. In this context, it is noteworthy that proopiomelanocortin (POMC) cleavage is also processed by furin, leading to ACTH, β-lipotropin, and β-endorphin. All prohormone convertases including furin are regulated by Ca2+. Because numerous epidermal peptides and enzymes are affected by H2O2-mediated oxidation, including the POMC-derived peptides α-MSH and β-endorphin as shown in the epidermis of patients with vitiligo, we here asked the question of whether furin could also be a possible target for this oxidation mechanism by using immunofluorescence, RT-PCR, Western blotting, Ca2+-binding studies, and computer modeling. Our results demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo (n = 10), suggesting H2O2-mediated oxidation. This was confirmed by 45Ca2+-binding studies with human recombinant furin identifying the loss of one Ca2+-binding site from the enzyme after oxidation with H2O2. Computer simulation supported alteration of one of the two Ca2+-binding sites on furin. Taken together, our results implicate that the Ca2+-dependent proteolytic activity of this convertase is targeted by H2O2, which in turn could contribute to the reduced epidermal expression of the POMC-derived peptides α-MSH and β-endorphin as documented earlier in patients with vitiligo.
32
Kappert, Kai, Vesna Furundzija, Jan Fritzsche, Christian Margeta, Janine Krüger, Heike Meyborg, Eckart Fleck, and Philipp Stawowy. "Integrin cleavage regulates bidirectional signalling in vascular smooth muscle cells." Thrombosis and Haemostasis 103, no. 03 (2010): 556–63. http://dx.doi.org/10.1160/th09-07-0478.
Full textAbstract:
SummaryIntegrins link the cytoskeleton to the extracellular matrix, providing outside-in/inside-out signalling essential for vascular smooth muscle cell (VSMC) migration in atherosclerosis. The integrin αv subunit is synthesised from its precursor via furin-dependent endoproteolytic cleavage. Furin is a proprotein convertase (PC) highly expressed in VSMCs and in human atherosclerotic lesions. Inhibition of αv processing inhibits binding to vitronectin and migration. However, the precise role of furin-dependent αv cleavage on integrin bidirectional signalling and subsequent VSMC functions is unknown. Our present study demonstrates that the furin-like PC inhibitor decanoyl-RVKR-chloromethylke-tone (dec-CMK) inhibited αv cleavage. This reduced vitronectin-induced (outside-in) focal adhesion kinase (FAK)- and paxillin-phosphorylation, and VSMC motility. Inside-out-stimulated, integrin-mediated VSMC adhesion/migration relied on integrin-adaptor protein activation following protein kinase C (PKC) and ERK1/2 phosphorylation. In contrast to outside-in signalling, PKC-dependent phosphorylation of FAK and paxillin was unaffected by the status of integrin cleavage. Still, cytoskeleton and focal adhesion site rearrangements were modulated by the inhibition of furin-dependent integrin cleavage, thereby lessening inside-out dependent migration. Hence, we find that integrin bidirectional signalling is critically controlled by furin. Furin-dependent integrin processing modulates rapid adaptive integrin/cytoskeleton changes, essential to VSMC motility, which represents a crucial component in atherosclerosis and restenosis.
33
Hipp, Madeleine, Uzi Gileadi, Dawn Sheperd, Caetano Reis e Sousa, and Vincenzo Cerundolo. "Processing of human TLR7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. (P1244)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 138.20. http://dx.doi.org/10.4049/jimmunol.190.supp.138.20.
Full textAbstract:
Abstract Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes. Although this mechanism underscores the ability of TLR7 to distinguish between self and not self RNA, the biosynthetic pathway of TLR7 remains unclear. Here, we show for the first time that human TLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments, where it is functionally active. Human TLR7 processing depends on the action of furin-like proprotein convertases and occurs at neutral pH. Knockdown of Furin and mutation of a furin-like recognition site within TLR7 inhibits processing of the receptor. Furthermore pro-inflammatory stimuli such as PMA and IFN-γ enhance processing of and signalling through TLR7, consistent with an observed upregulation of Furin. This mechanism might serve as a positive feedback loop generating active TLR7 after infection. As self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like proprotein convertases as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.
34
Keil, Günther M., Constanze Höhle, Katrin Giesow, and Patricia König. "Engineering Glycoprotein B of Bovine Herpesvirus 1 To Function as Transporter for Secreted Proteins: a New Protein Expression Approach." Journal of Virology 79, no. 2 (January 15, 2005): 791–99. http://dx.doi.org/10.1128/jvi.79.2.791-799.2005.
Full textAbstract:
ABSTRACT Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. König, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-α) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-α, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-α was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-α, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-α fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems.
35
Duda, Anja, Annett Stange, Daniel Lüftenegger, Nicole Stanke, Dana Westphal, Thomas Pietschmann, Scott W. Eastman, Maxine L. Linial, Axel Rethwilm, and Dirk Lindemann. "Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Processing Is Mediated by a Furin-Like Cellular Protease, but Cleavage Is Not Essential for Viral Infectivity." Journal of Virology 78, no. 24 (December 15, 2004): 13865–70. http://dx.doi.org/10.1128/jvi.78.24.13865-13870.2004.
Full textAbstract:
ABSTRACT Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.
36
Wool-Lewis, Rouven J., and Paul Bates. "Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function." Journal of Virology 73, no. 2 (February 1, 1999): 1419–26. http://dx.doi.org/10.1128/jvi.73.2.1419-1426.1999.
Full textAbstract:
ABSTRACT Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP1 and GP2, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.
37
PLAIMAUER, Barbara, Gabriele MOHR, Waltraud WERNHART, Michèle HIMMELSPACH, Friedrich DORNER, and Uwe SCHLOKAT. "‘Shed’ furin: mapping of the cleavage determinants and identification of its C-terminus." Biochemical Journal 354, no. 3 (March 8, 2001): 689–95. http://dx.doi.org/10.1042/bj3540689.
Full textAbstract:
The human endoprotease furin is involved in the proteolytic maturation of the precursor molecules of a wide variety of bioactive proteins. Despite its localization in the membranes of the trans-Golgi system by means of a transmembrane domain, it has repeatedly been reported to form a C-terminally truncated, naturally secreted form referred to as ‘shed’ furin. In order to identify the cleavage site, internal deletion mutants of increasing size, N-terminal to Leu708, and subsequently individual amino acid substitutions were introduced, and Arg683 was identified as the prime determinant for shedding. MS analysis determined Ser682 as the C-terminus of shed furin, suggesting that monobasic cleavage may occur N-terminal to Arg683. Alteration of Arg683 directs the shedding mechanism to alternative cleaving sites previously unused.
38
Carattino, Marcelo D., Gunhild M. Mueller, Lawrence G. Palmer, Gustavo Frindt, Anna C. Rued, Rebecca P. Hughey, and Thomas R. Kleyman. "Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease." American Journal of Physiology-Renal Physiology 307, no. 9 (November 1, 2014): F1080—F1087. http://dx.doi.org/10.1152/ajprenal.00157.2014.
Full textAbstract:
During maturation, the α- and γ-subunits of the epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143 and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186 site. We generated a polyclonal antibody that recognizes the region 144–186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186 cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αβHA-γ-V5 channels, but only the full-length (93 kDa) γ-subunit from oocytes expressing αβHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+ diet. We found that a low-Na+ diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+ diet.
39
Bronnimann, Matthew P., Christine M. Calton, Samantha F. Chiquette, Shuaizhi Li, Mingfeng Lu, Janice A. Chapman, Kristin N. Bratton, Angela M. Schlegel, and Samuel K. Campos. "Furin Cleavage of L2 during Papillomavirus Infection: Minimal Dependence on Cyclophilins." Journal of Virology 90, no. 14 (April 27, 2016): 6224–34. http://dx.doi.org/10.1128/jvi.00038-16.
Full textAbstract:
ABSTRACTDespite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, thePropionibacterium shermaniitranscarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.IMPORTANCEFurin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.
40
Khodarovich, Yu M., E. V. Konovalova, A. A. Schulga, S. M. Deyev, and R. V. Petrov. "Removal of the translocation domain and the furin cleavage site decreases the relative hepatotoxicity of the targeted antitumor toxins." Доклады Академии наук 489, no. 2 (November 20, 2019): 209–12. http://dx.doi.org/10.31857/s0869-56524892209-212.
Full textAbstract:
Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatoto-xicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.
41
BISSONNETTE, Lyne, Gabriel CHAREST, Jean-Michel LONGPRÉ, Pierre LAVIGNE, and Richard LEDUC. "Identification of furin pro-region determinants involved in folding and activation." Biochemical Journal 379, no. 3 (May 1, 2004): 757–63. http://dx.doi.org/10.1042/bj20031902.
Full textAbstract:
The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans-Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's pro-region.
42
Bestle, Dorothea, Miriam Ruth Heindl, Hannah Limburg, Thuy Van Lam van, Oliver Pilgram, Hong Moulton, David A. Stein, et al. "TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells." Life Science Alliance 3, no. 9 (July 23, 2020): e202000786. http://dx.doi.org/10.26508/lsa.202000786.
Full textAbstract:
The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2′ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
43
Klimstra, William B., Natasha L. Tilston-Lunel, Sham Nambulli, James Boslett, Cynthia M. McMillen, Theron Gilliland, Matthew D. Dunn, et al. "SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients." Journal of General Virology 101, no. 11 (November 1, 2020): 1156–69. http://dx.doi.org/10.1099/jgv.0.001481.
Full textAbstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.
44
Negahdaripour, Manica, Mohammad Reza Rahbar, Zahra Mosalanejad, and Ahmad Gholami. "Theta-Defensins to Counter COVID-19 as Furin Inhibitors: In Silico Efficiency Prediction and Novel Compound Design." Computational and Mathematical Methods in Medicine 2022 (February 9, 2022): 1–15. http://dx.doi.org/10.1155/2022/9735626.
Full textAbstract:
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was characterized as a pandemic by the World Health Organization (WHO) in Dec. 2019. SARS-CoV-2 binds to the cell membrane through spike proteins on its surface and infects the cell. Furin, a host-cell enzyme, possesses a binding site for the spike protein. Thus, molecules that block furin could potentially be a therapeutic solution. Defensins are antimicrobial peptides that can hypothetically inhibit furin because of their arginine-rich structure. Theta-defensins, a subclass of defensins, have attracted attention as drug candidates due to their small size, unique structure, and involvement in several defense mechanisms. Theta-defensins could be a potential treatment for COVID-19 through furin inhibition and an anti-inflammatory mechanism. Note that inflammatory events are a significant and deadly condition that could happen at the later stages of COVID-19 infection. Here, the potential of theta-defensins against SARS-CoV-2 infection was investigated through in silico approaches. Based on docking analysis results, theta-defensins can function as furin inhibitors. Additionally, a novel candidate peptide against COVID-19 with optimal properties regarding antigenicity, stability, electrostatic potential, and binding strength was proposed. Further in vitro/in vivo investigations could verify the efficiency of the designed novel peptide.
45
Laprise, Marie-Hélène, Francine Grondin, Pauline Cayer, Patrick P. McDonald, and Claire M. Dubois. "Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates." Blood 100, no. 10 (November 15, 2002): 3578–87. http://dx.doi.org/10.1182/blood.v100.10.3578.
Full textAbstract:
The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate–induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC–promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1–regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions −66 and +62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1–stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor α1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-β1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed forfur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions.
46
Wiens, Mayim E., and Jason G. Smith. "Alpha-Defensin HD5 Inhibits Furin Cleavage of Human Papillomavirus 16 L2 To Block Infection." Journal of Virology 89, no. 5 (December 24, 2014): 2866–74. http://dx.doi.org/10.1128/jvi.02901-14.
Full textAbstract:
ABSTRACTHuman papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human α-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data support a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid.IMPORTANCEOur study elucidates a new antiviral action for α-defensins against nonenveloped viruses in which HD5 directly interferes with a critical host-mediated viral processing step, furin cleavage of L2, at the cell surface. Blocking this key event has deleterious effects on the intracellular steps of virus infection. Thus, in addition to informing the antiviral mechanisms of α-defensins, our studies highlight the critical role of furin cleavage in HPV entry. Innate immune control, mediated in part by α-defensins expressed in the genital mucosa, may influence susceptibility to HPV infections that lead to cervical cancer. Moreover, understanding the mechanism of these natural antivirals may inform the design of therapeutics to limit HPV infection.
47
Roy, Sourav, Prithwi Ghosh, Abhirup Bandyopadhyay, and Sankar Basu. "Capturing a Crucial ‘Disorder-to-Order Transition’ at the Heart of the Coronavirus Molecular Pathology—Triggered by Highly Persistent, Interchangeable Salt-Bridges." Vaccines 10, no. 2 (February 16, 2022): 301. http://dx.doi.org/10.3390/vaccines10020301.
Full textAbstract:
The COVID-19 origin debate has greatly been influenced by genome comparison studies of late, revealing the emergence of the Furin-like cleavage site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a ‘disorder-to-order transition’ of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike–Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seem to be the other key characteristic features of the Spike–Furin binding in SARS-CoV-2, allowing the system to breathe—a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike–Furin binding events, generally, in the coronavirus lineage, seems to have preference for ionic bond formation, even with a lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of characteristic metastabilities attributed to Spike–Furin interactions, generally to the coronavirus lineage, which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. The current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution, but also add substantially to the existing theories of proteolytic cleavages.
48
Jean, F., A. Basak, J. DiMaio, N. G. Seidah, and C. Lazure. "An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor." Biochemical Journal 307, no. 3 (May 1, 1995): 689–95. http://dx.doi.org/10.1042/bj3070689.
Full textAbstract:
Based upon the observed cleavage of various peptidyl substrates by the recombinant prohormone convertases PC1 and furin, an intramolecularly quenched fluorogenic peptidyl substrate, (o-aminobenzoyl)-Lys-Glu-Arg-Ser-Lys-Arg-Ser-Ala-Leu-Arg-Asp-(3-nitro)Ty r-Ala, was synthesized. In spite of the distance (approx. 33 A) separating the fluorescent donor/acceptor pair, the highly fluorescent o-aminobenzoyl group is efficiently quenched by long-range resonance energy transfer to the (3-nitro)Tyr moiety. Both recombinant human PC1 and human furin recognize and cleave specifically this substrate at the expected Arg-Ser site in a sensitive manner. The Km values for human PC1 and human furin were 17 microM and 30 microM respectively, with Vmax. values of 6.4 microM/h and 18 microM/h. These values differ significantly from those obtained when using a 7-amino-4-methylcoumarin-containing pentapeptidyl substrate where, for similar Km values, the Vmax. values were much lower. The peptide sequence was used to synthesize another peptide incorporating a ketomethylene arginyl pseudopeptide bond. This compound proved to be a potent competitive inhibitor of both human PC1 and human furin, displaying Ki values of 7.2 microM and 2.4 microM respectively.
49
Neumann, Gabriele, Heinz Feldmann, Shinji Watanabe, Igor Lukashevich, and Yoshihiro Kawaoka. "Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture." Journal of Virology 76, no. 1 (January 1, 2002): 406–10. http://dx.doi.org/10.1128/jvi.76.1.406-410.2002.
Full textAbstract:
ABSTRACT Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.
50
Johnson, Bryan A., Xuping Xie, Adam L. Bailey, Birte Kalveram, Kumari G. Lokugamage, Antonio Muruato, Jing Zou, et al. "Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis." Nature 591, no. 7849 (January 25, 2021): 293–99. http://dx.doi.org/10.1038/s41586-021-03237-4.
Full text