Biology subjects

Seidah, N. G.

Publications and source records attributed to Seidah, N. G..

6 recordsLinked to original sources

bioRxiv2025

Proteolytic processing of the Marburg virus glycoprotein depends on Sec616β and is required for cell entry

Ebola and Marburg virus (EBOV, MARV) cause severe disease and therapeutic options are urgently needed. The Sec61 translocon facilitates ER import of viral glycoproteins (GPs) and may represent a therapeutic target. Here, we report that the Sec61 subunit Sec61{beta}, although dispensable for GP expression, is required for proteolytic cleavage of MARV- but not EBOV-GP and that an intact furin motif is essential for robust cell entry of Marburg- but not Ebolaviruses. Further, MARV- but not EBOV-GP was cleaved by the furin-related enzyme SKI-1, for which a cleavage motif was identified in silico, and cleavage by SKI-1 was impaired in SEC61B-KO cells. In addition, Sec61{beta} was required for normal N-glycosylation of MARV-GP and mutation of a sequon (N563D) abrogated cleavage. Finally, the absence of Sec61{beta} modestly, and blockade of Sec61 via apratoxin S4 markedly, inhibited EBOV and MARV infection. These results reveal a differential protease dependence of MARV and EBOV and identify Sec61 as a potential therapeutic target. Author summaryThe filoviruses Ebola virus (EBOV) and Marburg virus (MARV) spread from animals to humans and can cause deadly outbreaks. These viruses rely on a surface glycoprotein (GP) for infection, which is processed by the enzyme furin in infected human cells. Cleavage of EBOV-GP was thought to be non-essential for infection. However, using lab models for filovirus entry into cells, we discovered that MARV, unlike EBOV, needs this cleavage step to infect cells efficiently. We also found that the host cell protein Sec61{beta} is necessary for proteolytic processing and glycosylation of MARV-GP but not EBOV-GP. In addition, we showed that another cellular enzyme, SKI-1, can process MARV- but not EBOV-GP. Finally, we found that removing Sec61{beta} or blocking Sec61 activity reduced infection by both viruses. These findings show key differences in how the two viruses interact with host cells and suggest that targeting Sec61 could be a promising new strategy to fight Ebola and Marburg virus infections.

microbiology↗

bioRxiv2023

Intermolecular proteolytic processing of SPRING and Site-1-protease regulate SREBP signaling

The SREBP transcription factors are central regulators of fatty acid and cholesterol metabolism. Produced as membrane-resident precursor proteins in the ER, their transcriptional activation requires the cholesterol-dependent translocation to the Golgi, and subsequent proteolytic cleavage by S1P, a type-I transmembrane protein. S1P is produced as a proprotein convertase that needs to undergo autocatalytic cleavage to attain its mature form in the Golgi, in a process that is not fully elucidated. We have recently identified SPRING (C12ORF49) as a novel regulator of the SREBP pathway and reported that S1P activity and retrograde recycling of the SREBP chaperone SCAP are affected. Here, we demonstrate that SPRING and S1P interact and that in co-transfection experiments in mammalian cells this facilitates the autocatalytic activation of S1PA[->]C form. Accordingly, S1PA[->]C processing of stably overexpressed S1P in SPRINGKO cells is attenuated, but not abolished, and does not rescue SREBP signaling. Reciprocally, we identified a conserved S1P cleavage site in SPRING, and demonstrate that cleavage of SPRING results in secretion of the SPRING ectodomain. SPRING cleavage is S1P-specific and can be pharmacologically inhibited by S1P inhibitors or by mutating the S1P cleavage site. Functional analysis revealed that the SPRING ectodomain was sufficient to support S1PA[->]C processing and SREBP signaling, but that SPRING cleavage is not a prerequisite for this. In conclusion, our study reveals a complex interplay between the proteolytic activation of S1P and SPRING yet suggests that this is not the primary mechanism underlying the role of SPRING in SREBP signaling.

biochemistry↗

bioRxiv2021

Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2' by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2

Disclaimer StatementThe author has withdrawn this manuscript due to a duplicate posting of manuscript number 423106. Therefore, the author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author (Nabil G. Seidah at seidahn@ircm.qc.ca.

biochemistry↗

bioRxiv2021

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLRthat is shed by Furin

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote-carriers of ASGR1-deletions exhibited [~]34% lower risk of coronary artery disease and [~]10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, the regulation of LDLR and/or PCSK9 by ASGR1 was studied. Thus, we investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality by Western-blot and immunofluorescence in HepG2 naive and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. Such lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in HepG2 naive cells significantly increased total ([~]1.2-fold) and cell-surface ([~]4-fold) LDLR protein. In HepG2-PCSK9-knockout cells ASGR1-silencing led to [~]2-fold higher levels of LDLR protein and DiI-LDL uptake, associated with [~]9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 reduced primarily the immature non-O-glycosylated LDLR ([~]110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR ([~]150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103{downarrow} into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.

biochemistry↗

bioRxiv2021

Genetic analysis of the contribution of the proprotein convertases to the in vivo regulation of FGF23

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179{downarrow}, but whether they are fulfilling this function in vivo is currently unknown. To address this question we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1{beta} injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

physiology↗

bioRxiv2018

A novel cell-based sensor detecting the activity of individual basic proprotein convertases

The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides and do not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. We report here PCific, a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. PCific consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in cis. Screening of selected candidate peptidomimetic inhibitors revealed that PCific can readily distinguish between general and selective PC inhibitors in a high-throughput screening format.

biochemistry↗