Biomedical subjects

Nabil G Seidah

Publications and source records attributed to Nabil G Seidah.

At least 19 recordsLinked to original sources

PubMed2007

Three common alleles of KIR2DL4 (CD158d) encode constitutively expressed, inducible and secreted receptors in NK cells.

Genetic polymorphism of KIR2DL4 results in alleles with either 9 or 10 consecutive adenines in exon 6, which encodes the transmembrane domain. "10A" alleles encode a membrane-expressed receptor that is constitutively expressed on resting CD56bright NK cells and on CD56dim cells after culture. However, in some individuals with the 10A allele, KIR2DL4 cannot be detected on their resting CD56bright NK cells. "9A" alleles have been predicted to encode a secreted receptor due to the splicing out of the transmembrane region. In this publication, we show that those individuals with a 10A allele who lack detectable KIR2DL4 on CD56bright NK cells express a KIR2DL4 receptor in which the D0-domain is excised. This Delta-D0 receptor cannot be detected by the available anti-KIR2DL4 monoclonal antibodies. In such individuals, KIR2DL4 becomes detectable on cultured NK cells due to up-regulation of the full-length KIR2DL4 transcript. In all individuals with 10A alleles, KIR2DL4 ceases to be expressed at the cell surface 16 days after activation, despite the maintenance of maximal levels of KIR2DL4 mRNA transcription, suggesting the existence of a negative regulator of cell surface expression. Finally, we show that the 9A allele can produce a secreted KIR2DL4 receptor.

Alleles↗

PubMed2006

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation.

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.

Animals↗

PubMed2006

The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications.

PCSK9 is the ninth member of the proprotein convertase (PC) family. Some of its natural mutations have been genetically associated with the development of a dominant form of familial hyper- or hypocholesterolemia. The exact mechanism of action of PCSK9 is not clear, although it is known to enhance the intracellular degradation of the low density lipoprotein (LDL) receptor in acidic compartments, likely the endosomes/lysosomes. We analyzed the post-translational modifications of PCSK9 and show that it is sulfated within its prosegment at Tyr38. We also examined the susceptibility of PCSK9 to proteolytic cleavage by the other members of the PC family. The data show that the natural gain-of-function mutations R218S, F216L, and D374Y associated with hypercholesterolemia result in total or partial loss of furin/PC5/6A processing at the motif RFHR218 downward arrow. In contrast, the loss-of-function mutations A443T and C679X lead either to the lack of trans-Golgi network/recycling endosome localization and an enhanced susceptibility to furin cleavage (A443T) or to the inability of PCSK9 to exit the endoplasmic reticulum (C679X). Furthermore, we report the presence of both native and furin-like cleaved forms of PCSK9 in circulating human plasma. Thus, we propose that PCSK9 levels are finely regulated by the basic amino acid convertases furin and PC5/6A. The latter may reduce the lifetime of this proteinase and its ability to degrade the cell-surface LDL receptor, thereby regulating the levels of circulating LDL cholesterol.

Amino Acid Sequence↗

PubMed2006

Increased stress-induced analgesia in mice lacking the proneuropeptide convertase PC2.

Many neuropeptides involved in pain perception are generated by endoproteolytic cleavages of their precursor proteins by the proprotein convertases PC1 and PC2. To investigate the role of PC2 in nociception and analgesia, we tested wild-type and PC2-null mice for their responses to mechanical and thermal nociceptive stimuli, before and after a short swim in cold or warm water. Basal responses and responses after a cold swim were similar between the two groups. However, after a short forced swim in warm water, PC2-null mice were significantly less responsive to the stimuli than wild-type mice, an indication of increased opioid-mediated stress-induced analgesia. The enhanced analgesia in PC2-null mice may be caused by an accumulation of opioid precursor processing intermediates with potent analgesic effects, or by loss of anti-opioid peptides.

Animals↗

PubMed2006

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Amino Acid Sequence↗

PubMed2006

Unexpected similarity between the cytosolic West Nile virus NS3 and the secretory furin-like serine proteinases.

Many viral proteins undergo proteolytic processing events that are required for virus infection and virion assembly. In this issue of Biochemical Journal, Strongin and co-workers report that the NS3 protease from West Nile virus unexpectedly cleaves certain substrates at pairs of basic residues, a specificity that resembles that of the furin-like PCs (proprotein convertases). This led to the demonstration that furin/PC inhibitors containing poly(D-arginine) are also potent inhibitors of NS3, and that anthrax toxin protective antigen and myelin basic protein are potential NS3 substrates. Structural modelling based on Dengue virus NS3 provided a possible rationale for the observed cleavage specificity of West Nile virus NS3.

Amino Acid Sequence↗

PubMed2006

Proprotein convertases: lessons from knockouts.

The physiological role of the subtilisin/kexin-like proprotein convertases (PCs) in rodents has been examined through the use of knockout mice. This review will summarize the major in vivo defects that result from the disruption of the expression of their genes. This includes abnormal embryonic development, hormonal disorder, infertility, and/or modified lipid/sterol metabolism. Members of the PC family play a central role in the processing of various protein precursors ranging from hormones and growth factors to bacterial toxins and viral glycoproteins. Proteolysis occurring at basic residues is mediated by the basic amino acid-specific proprotein convertases, namely: PC1/3, PC2, furin, PACE4, PC4, PC5/6, and PC7. In contrast, proteolysis at nonbasic residues is performed by the subtilisin/kexin-like isozyme-1 (SKI-1/S1P) and the newly identified neural apoptosis-regulated convertase-1 (PCSK9/NARC-1). In addition to their requirement for many physiological processes, these enzymes are also involved in various pathologies such as cancer, obesity, diabetes, lipid disorders, infectious diseases, atherosclerosis and neurodegenerative diseases.

Animals↗

PubMed2006

Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system.

Neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low-density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT-PCR analyses were performed following retinoic acid (RA)-induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC-1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element-binding protein-2, which normally up-regulates NARC-1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC-1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole-mount in situ hybridization revealed that the pattern of expression of zebrafish NARC-1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC-1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain-midbrain boundaries, leading to embryonic death at approximately 96 h after fertilization. These data support a novel role for NARC-1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.

Animals↗

PubMed2006

Deletion of the gene encoding proprotein convertase 5/6 causes early embryonic lethality in the mouse.

PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5(Delta4)-deficient allele missing exon 4 that encodes the catalytic Asp173. While Delta4/+ heterozygotes were healthy and fertile, genotyping of progeny obtained from Delta4/+ interbreeding indicated that Delta4/Delta4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene.

Adrenal Cortex↗

PubMed2006

The proprotein convertases and their implication in sterol and/or lipid metabolism.

The proprotein convertases represent a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7, and two other subtilases that cleave at non-basic residues, called SKI-1/S1P and NARC-1/PCSK9. The present review concentrates on the regulatory role played by some of these convertases in cholesterol and lipid metabolism. Thus, PC5/6, PACE4 and Furin upregulate high-density lipoprotein (HDL) levels via the inactivation of endothelial and lipoprotein lipases. The SKI-1/S1P-directed cleavage of membrane-bound transcription factors known as sterol regulatory element binding proteins (SREBP-1 and SREBP-2) results in upregulation of the synthesis of sterols, lipids and the LDL receptor (LDLR). Finally, PCSK9 downregulates the protein levels of the LDLR by enhancement of its intracellular metabolic pathway in subcellular acidic compartments.

Amino Acid Sequence↗

PubMed2005

Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi.

Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them.

Amino Acid Sequence↗

PubMed2005

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

Animals↗

PubMed2005

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread.

BACKGROUND: Severe acute respiratory syndrome (SARS) is caused by a newly discovered coronavirus (SARS-CoV). No effective prophylactic or post-exposure therapy is currently available. RESULTS: We report, however, that chloroquine has strong antiviral effects on SARS-CoV infection of primate cells. These inhibitory effects are observed when the cells are treated with the drug either before or after exposure to the virus, suggesting both prophylactic and therapeutic advantage. In addition to the well-known functions of chloroquine such as elevations of endosomal pH, the drug appears to interfere with terminal glycosylation of the cellular receptor, angiotensin-converting enzyme 2. This may negatively influence the virus-receptor binding and abrogate the infection, with further ramifications by the elevation of vesicular pH, resulting in the inhibition of infection and spread of SARS CoV at clinically admissible concentrations. CONCLUSION: Chloroquine is effective in preventing the spread of SARS CoV in cell culture. Favorable inhibition of virus spread was observed when the cells were either treated with chloroquine prior to or after SARS CoV infection. In addition, the indirect immunofluorescence assay described herein represents a simple and rapid method for screening SARS-CoV antiviral compounds.

Ammonium Chloride↗

PubMed2005

Proprotein convertases [corrected] are responsible for proteolysis and inactivation of endothelial lipase.

Plasma lipoprotein metabolism is tightly regulated by several members of the triglyceride lipase family, including endothelial lipase (EL) and lipoprotein lipase (LPL). Our previous work suggested that EL is proteolytically processed. In this report, we have used a combination of epitope tagging, mutagenesis, and N-terminal sequencing to determine the precise location of the cleavage site within EL. The cleavage occurs immediately after the sequence RNKR, a known recognition sequence for the proprotein convertase (PC) family. We demonstrate that some PCs, but not all, can proteolytically cleave EL at this site and thereby directly regulate EL enzymatic activity through modulating EL cleavage. Furthermore, specific knockdown of individual PCs proves that PCs are the proteases that cleave EL in human endothelial cells. Interestingly, a homologous site in LPL is also cleaved by PCs. This action is unusual for PCs, which are traditionally known as activators of pro-proteins, and highlights a potential role of PCs in lipid metabolism through their proteolytic processing of lipases.

Blotting, Western↗

PubMed2005

Furin-like proprotein convertases are central regulators of the membrane type matrix metalloproteinase-pro-matrix metalloproteinase-2 proteolytic cascade in atherosclerosis.

BACKGROUND: Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. METHODS AND RESULTS: Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-alpha or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP-dependent activation of vascular smooth muscle cell-derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. CONCLUSIONS: Furin-like PCs (furin and PC5) play a central role in a MT-MMP-MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Arteriosclerosis↗

PubMed2005

Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.

Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-beta, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies.

Carcinoma, Squamous Cell↗

PubMed2005

Immunohistochemical localization of subtilisin/kexin-like proprotein convertases in human atherosclerosis.

Integrins are heterodimeric alpha/beta receptors that link the cytoskeleton with the extracellular matrix, thereby regulating several cell functions important in atherosclerosis. In vitro, the subtilisin/kexin-like proprotein convertases (PCs), namely PC5 and furin, have been shown to be responsible for the endoproteolytic activation of the alpha(v) integrin subunit. Based on their cleavage activity, these PCs are potential targets in atherosclerosis. In the present study, we investigated the localization of furin and PC5 in different stages of human atherosclerosis. Immunohistochemical analysis of furin and PC5 revealed their presence in vascular smooth-muscle cells and endothelial cells in atherosclerotic and non-atherosclerotic lesions. However, in the more advanced lesions, furin and PC5 staining was significantly expressed in macrophages/foam cells. In vitro, THP-1 derived macrophages contained furin and PC5, and maturation of monocytes to macrophages was accompanied by enhanced alpha(v)beta3 cell-surface expression. Inhibition of furin/PC5 with the specific pharmacological furin-like PC-inhibitor dec-CMK inhibited alpha(v) endoproteolytic activation but did not abolish alpha(v)beta3 cell-surface expression. This indicates that furin/PC5 is required for alpha(v) endoproteolytic activation but not for alpha(v) routing and sorting to the cell surface. In conclusion, our study demonstrates that furin and PC5 are significantly expressed in mononuclear cells in advanced human atherosclerotic lesions, where they regulate alpha(v) endoproteolytic activation.

Arteriosclerosis↗

PubMed2005

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells.

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.

Adenoviridae↗