Furin

The virus responsible for the COVID-19 global health crisis, SARS-CoV-2, has been shown to utilize the ACE2 protein as an entry point to its target cells. The virus has been shown to rely on the actions of TMPRSS2 (a serine protease), as well as FURIN (a peptidase), for the critical priming of its spike protein. It has been postulated that variations in the sequence and expression of SARS-CoV-2’s receptor (ACE2) and the two priming proteases (TMPRSS2 and FURIN) may be critical in contributing to SARS-CoV-2 infectivity. This study aims to examine the different expression levels of FURIN in various tissues and age ranges in light of ACE2 and TMPRSS2 expression levels using the LungMAP database. Furthermore, we retrieved expression quantitative trait loci (eQTLs) of the three genes and their annotation. We analyzed the frequency of the retrieved variants in data from various populations and compared it to the Egyptian population. We highlight FURIN’s potential interplay with the immune...

RPE. 40 mutant cells differ from wild-type Chinese hamster ovary (CHO-K1) cells in their increased resistance toPseudomonas exotoxin A and their inability to process the insulin proreceptor and certain viral envelope proproteins. Northern analysis revealed that RPE. 40 cells maintained a substantially lower steady-state level of 4.0 kbfur mRNA than did CHO-K1 cells. Analysis offur cDNAs showed that RPE. 40 cells were diploid at thefur locus, and RPE. 40 cells had a Cys (TGC) to Tyr (TAC) mutation in codon 196 of one allele (allele I). Approximately 25–30% of the CHO-K1 cells were also heterozygous (Tyr/Cys) at codon 196, and pre-mRNAs transcribed from the second allele (allele II) in RPE. 40 cells were defectively spliced. All other pre-mRNAs were correctly spliced. Rapid turnover of defectively spliced transcripts may account for the reduced steady-state level offur mRNA observed in RPE. 40 cells. Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE. 40 cells.

Proprotein convertases mediate the production of a variety of peptidic mitogens by limited proteolysis of their precursors. These proteases may also participate in the autocrine production of such mitogens by cancer cells and thus contribute to the unchecked proliferation of these cells. As a step towards defining this contribution, we have examined the levels of four convertase mRNAs in human lung neoplasms using semiquantitative Northern blot analysis. Furin mRNA was expressed in all the tumours; its level in squamous cell carcinomas and adenocarcinomas was on average about threefold higher than in small-cell lung carcinomas (SCLCs). PACE4 transcripts were detected in eight of 14 adenocarcinomas and in seven of 17 squamous cell carcinomas; they were detectable in only two of seven SCLCs. PC1 mRNA was undetected in squamous cell carcinomas and in all but two adenocarcinomas; it was present in four of six SCLCs. PC2 mRNA was found in two adenocarcinomas, in one squamous cell carcino...

Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment.

Infectious HIV-1 requires gp160 cleavage by furin at the REKR511↓ motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Improvement of treatment efficacy and decreased side effects through tumor-targeted drug delivery would be desirable. By panning with a phage-displayed cyclic random peptide library we ...

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and ce...

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

Matrix metalloproteinases (MMPs) disrupt the blood—brain barrier (BBB) during reperfusion. Occludin and claudins are recently described tight junction proteins (TJPs) that form the BBB. We hypothesized that the opening of the BBB was because of the degradation of TJPs by the MMPs. Spontaneously hypertensive rats had a 90 mins middle cerebral artery occlusion with reperfusion for 2, 3, or 24 h. Matrix metalloproteinases were measured by immunohistochemistry and in situ and gel zymography. Real-time polymerase chain reaction (PCR) measured mRNAs of MMP-2 and −9, furin, membrane-type MMP (MT1-MMP), occludin, and claudin-5. There was opening of the BBB in the piriform cortex after 3 h of reperfusion, and an MMP inhibitor, BB-1101 (30 mg/kg), prevented the opening. At 3 h, in situ zymograms showed gelatinase activity. Zymography and PCR showed greater increases in MMP-2 than in MMP-9. There were increased mRNA and immunohistochemistry for MT1-MMP and furin, which activate MMP-2. Claudin-...

Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R(↓)R-ACC and Z-R(↓)R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 ...

β-1,3-Glucanases (E.C. 3.2.1.39) are widely distributed enzyme among bacteria, fungi, and higher plants. Analyses of accumulation levels of β-1,3-glucanase protein in various tissues in banana have clearly indicated abundance of β-1,3-glucanase protein accumulation in ripe pulp tissue. After cloning of β-1,3-glucanase from banana pulp (cultivar Cavendish), we have carried out an in silico analysis to investigate the sequential, structural, and phylogenetic characteristics of the putative banana β-1,3-glucanase protein. As like other ripening specific genes, β-1,3-glucanase is regulated in response to a wide variety of factors. Therefore, we have analyzed the transcript accumulation pattern and protein levels of β-1,3-glucanase in response to ethylene, auxin, ABA, wounding and, low temperature in preclimacteric banana fruit. Expression profile analyses have indicated that whereas exogenous application of ethylene strongly stimulated β-1,3-glucanase transcript accumulation, ABA partially induced the expression of the gene. On the other hand, wound treatment did not induce β-1,3-glucanase expression. Conversely, auxin and cold treatment negatively regulated β-1,3-glucanase gene expression and thus inhibited glucanase activity. In addition, β-1,3-glucanase transcript level was markedly decreased by constant exposure to white light. Protein level and enzymatic activity of β-1,3-glucanase were substantially increased with considerable decrease in fruit firmness by ethylene treatment and reduced exposure to white light conditions as compared with other treatments. Together, the overall study of β-1,3-glucanase expression pattern, glucanase activity, and changes in fruit firmness during ripening in various conditions suggest the possible physiological function of β-1,3-glucanase in fruit pulp softening.

Furin-dependent maturation of the BRI2 protein generates the Bri2-23 fragment that is able to arrest the aggregation of amyloidβ, the peptide implicated in Alzheimer's disease (AD). Bri2-23 contains cysteines at positions 5 and 22, which are likely to bind to metal ions such as Cu(i). Metal ions may play a role in the etiology of neurodegenerative disorders such as AD, and in this work we explore the metal ion induced folding and aggregation of Bri2-23 using Hg(ii) and Ag(i) as spectroscopic probes with structural and ligand preferences similar to those of Cu(i), while not displaying redox activity under the experimental conditions. In general, interaction of Bri2-23 with soft metal ions changes the structural properties and solution behavior of the peptide that tune to increasing metal to peptide stoichiometry. Potentiometric, (199m)Hg PAC and ESI-MS data indicate that addition of up to 0.5 equivalents of Hg(ii) to Bri2-23 yields a two-coordinated HgS2 structure at the metal si...

ABSTRACTThe spîke (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic “priming” at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and “fusion-activation” at a S2’ site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2’, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2’ processing by Furin at KPSKR815↓ was strongly enhanced by ACE2, but not for the optimized S2’ KRRKR815↓ mutant (μS2’), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a “pH-dependent” endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a “pH-independent” viral en...

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg41↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg41XXXXArg46↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV inf...

Furin, the mammalian prototype of a family of serine proteases, is required for ventral closure and axial rotation, and formation of the yolk sac vasculature. Here we show additionally that left-sided expression of pitx2 and lefty-2 are also perturbed in Furin-deficient embryos. These tissue abnormalities are preceded by a marked delay in the expansion of the definitive endoderm during gastrulation. Using a chimera approach, we show that Furin activity is required in epiblast derivatives, including the primitive heart, gut and extraembryonic mesoderm, whereas it is nonessential in the visceral endoderm. Thus, chimeric embryos, derived by injecting wild-type embryonic stem (ES) cells into fur(-/-) blastocysts, develop normally until at least 9.5 d.p.c. In contrast, Furin-deficient chimeras developing in the context of wild-type visceral endoderm fail to undergo ventral closure, axial rotation and yolk sac vascularization. Fur(-/-) cells are recruited into all tissues examined, includ...

Mouse 3T3-L1 cells are widely used to study adipocyte differentiation in vitro. When treated with insulin, dexamethasone and isobutylmethylxanthine these fibroblastic cells differentiate into round triglyceride-rich adipocytes. Because several proteins implicated in adipocyte differentiation (e.g. type 1 IGF receptors) are proteolytically activated by endoproteinases of the proprotein convertase family, we sought to determine whether these endoproteinases are crucial for adipose conversion. In this study, we show that expression of the proprotein convertases PACE4, PC7 and furin increases when 3T3-L1 cells are induced to differentiate into adipocytes. The differentiation was blocked in transfected cells expressing alpha1-antitrypsin Portland or in normal cells pre-treated with the synthetic inhibitor decanoyl-RVKR-chloromethylketone. Both inhibitors are known to specifically inactivate proprotein convertases. The block was associated with impaired proteolytic activation of proIGF-1 ...