Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.
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PMID:The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity. 1056 53

We have produced and characterized, in a baculovirus expression system, simian-human immunodeficiency virus-like particles (SHIV VLPs) containing SIV Gag and HIV envelope (Env) proteins. Recombinant SIV gag (SIVmac239) and full-length or cytoplasmic domain-truncated HIV env from either HIV BH10 or HIV 89.6 virus were coexpressed in insect cells and Env incorporation into released SHIV VLPs was characterized. The expression level of the Env protein was found to be about 20-50% higher in both strains producing the truncated Env. Cell surface expression of the truncated Env proteins was found to be about eightfold higher than that of the full-length Env proteins. Furthermore, the truncated Env proteins exhibited higher levels of cleavage into gp120 and gp41 compared with the full-length Env. The SHIV VLPs produced by the coexpression of SIV gag and truncated HIV env contained both precursor (gp160) and gp120, while predominantly gp160 was found in the VLPs containing full-length Env. Coinfection of a recombinant virus expressing the protease furin also resulted in more efficient cleavage of gp160 to gp120. Both full-length and truncated Env were found to induce CD4+ cell fusion. Analysis of VLPs by immunoelectron microscopy demonstrated the incorporation of both full-length and truncated Env on the surface of VLPs. Truncated Env also was incorporated at higher levels on the surfaces of VLPs than full-length Env. The assembly of VLPs containing biologically active Env proteins may be useful in vaccine development and in functional studies of the HIV envelope protein.
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PMID:Production and characterization of simian--human immunodeficiency virus-like particles. 1071 Feb 11

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain, termed SOS gp140 (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variable-loop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.
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PMID:Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits. 1079 83

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/Delta41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and Deltagp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4- and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.
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PMID:Processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary HIV-1 isolate. 1092 31

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.
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PMID:Maturation of HIV envelope glycoprotein precursors by cellular endoproteases. 1106 80

PACS-1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV-1) NEF: We have now investigated the interaction of PACS-1 with heterotetrameric adaptor complexes. PACS-1 associates with both AP-1 and AP-3, but not AP-2, and forms a ternary complex between furin and AP-1. A short sequence within PACS-1 that is essential for binding to AP-1 has been identified. Mutation of this motif yielded a dominant-negative PACS-1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant-negative PACS-1 causes a mislocalization of both furin and mannose 6-phosphate receptor from the trans-Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant-negative PACS-1 inhibits the ability of HIV-1 Nef to downregulate MHC-I. These studies demonstrate the requirement for PACS-1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV-1 pathogenesis.
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PMID:PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic. 1133 85

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.
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PMID:Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins. 1186 26

PACS-1 is a cytosolic sorting protein that directs the localization of membrane proteins in the trans-Golgi network (TGN)/endosomal system. PACS-1 connects the clathrin adaptor AP-1 to acidic cluster sorting motifs contained in the cytoplasmic domain of cargo proteins such as furin, the cation-independent mannose-6-phosphate receptor and in viral proteins such as human immunodeficiency virus type 1 Nef. Here we show that an acidic cluster on PACS-1, which is highly similar to acidic cluster sorting motifs on cargo molecules, acts as an autoregulatory domain that controls PACS-1-directed sorting. Biochemical studies show that Ser278 adjacent to the acidic cluster is phosphorylated by CK2 and dephosphorylated by PP2A. Phosphorylation of Ser278 by CK2 or a Ser278-->Asp mutation increased the interaction between PACS-1 and cargo, whereas a Ser278-->Ala substitution decreased this interaction. Moreover, the Ser278-->Ala mutation yields a dominant-negative PACS-1 molecule that selectively blocks retrieval of PACS-1-regulated cargo molecules to the TGN. These results suggest that coordinated signaling events regulate transport within the TGN/endosomal system through the phosphorylation state of both cargo and the sorting machinery.
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PMID:The phosphorylation state of an autoregulatory domain controls PACS-1-directed protein traffic. 1463 83

Pro-hormone or pro-protein convertases are a conserved family of eukaryotic serine proteases found in the secretory pathway. These endoproteases mature precursors for peptides and proteins that perform a wide range of physiologically important and clinically relevant functions. The first member of this family to be identified was Kex2 in the yeast Saccharomyces cerevisiae. One mammalian member of this family - furin - is responsible for processing substrates that include insulin pro-receptor, human immunodeficiency virus gp160 glycoprotein, Ebola virus glycoprotein, and anthrax protective antigen. Recent determination of the crystal structures for the catalytic core domains of both Kex2 and furin - the first for any members of this family - provide remarkable insights and a new level of understanding of substrate specificity and catalysis by the pro-protein convertases.
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PMID:The kindest cuts of all: crystal structures of Kex2 and furin reveal secrets of precursor processing. 1510 34

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.
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PMID:Furin cleavage of the HIV-1 Tat protein. 1513 58


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