scholarly journals Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

1992 ◽  
Vol 3 (2) ◽  
pp. 143-155 ◽  
Author(s):  
D T Ng ◽  
S S Watowich ◽  
R A Lamb
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Complex Formation ◽  
Simian Virus ◽  
Single Site ◽  
Simian Virus 5 ◽  
Chaperone Protein ◽  
Protein Molecules ◽  
Mutant Polypeptides

The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated.

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase

1990 ◽  
Vol 10 (5) ◽  
pp. 1989-2001
Author(s):  
D T Ng ◽  
S W Hiebert ◽  
R A Lamb
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Carbohydrate Chain ◽  
Protein A ◽  
Simian Virus ◽  
Glycosylation Pattern ◽  
Transient Complex ◽  
Simian Virus 5 ◽  
Endoplasmic Reticulum Protein ◽  
Carbohydrate Chains

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.

scholarly journals The V Protein of Simian Virus 5 Inhibits Interferon Signalling by Targeting STAT1 for Proteasome-Mediated Degradation

1999 ◽  
Vol 73 (12) ◽  
pp. 9928-9933 ◽  
Author(s):  
L. Didcock ◽  
D. F. Young ◽  
S. Goodbourn ◽  
R. E. Randall
Keyword(s):  
Protein Synthesis ◽  
Defense Mechanisms ◽  
Simian Virus ◽  
Virus Protein ◽  
Structural Protein ◽  
V Protein ◽  
Antiviral State ◽  
Simian Virus 5 ◽  
Transcription Complexes

ABSTRACT To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the interferons (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor 3 and gamma-activated factor transcription complexes that are involved in activating IFN-α/β- and IFN-γ-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells

1988 ◽  
Vol 8 (9) ◽  
pp. 3740-3747
Author(s):  
H W Stürzbecher ◽  
C Addison ◽  
J R Jenkins
Keyword(s):  
Complex Formation ◽  
Protein Complexes ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mutant P53 ◽  
Cos Cells

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

scholarly journals High avidity cytotoxic T lymphocytes to a foreign antigen are efficiently activated following immunization with a recombinant paramyxovirus, simian virus 5

2002 ◽  
Vol 83 (5) ◽  
pp. 1167-1172 ◽  
Author(s):  
Griffith D. Parks ◽  
Martha A. Alexander-Miller
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Simian Virus ◽  
Effective Vaccine ◽  
High Avidity ◽  
Vaccine Vectors ◽  
Ctl Response ◽  
Foreign Antigen ◽  
Simian Virus 5

Our previous work has shown that high avidity cytotoxic T lymphocytes (CTL) are optimal for virus clearance in vivo and thus it is necessary that an effective vaccine is capable of eliciting high avidity CTL. To determine if vaccination with the paramyxovirus simian virus 5 (SV5) elicits a high avidity response to a model foreign antigen, a recombinant virus was engineered to express chicken ovalbumin (rSV5–Ova). To compare the CTL response elicited with rSV5–Ova and a recombinant vaccinia virus expressing ovalbumin (rVV–Ova), mice were vaccinated intranasally with various doses of each vector and the Ova-specific CTL response was determined by ELISPOT analysis. Here, it has been shown that rSV5 can be equally as effective as rVV in eliciting antigen-specific CTL, in terms of both the total number of CTL and the number of high avidity cells. This has implications for both the design of vaccine vectors and the route utilized for vaccine administration for the elicitation of high avidity CTL responses. The advantages and future potential use of rSV5 vaccine vectors are discussed.

scholarly journals Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

2020 ◽  
Author(s):  
Weirui Ma ◽  
Gang Zhen ◽  
Wei Xie ◽  
Christine Mayr
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Complex Formation ◽  
Major Site ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Protein Complex Formation ◽  
Unstructured Regions

SummaryThe TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are sphere-like, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3′UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.

scholarly journals rsly1 Binding to Syntaxin 5 Is Required for Endoplasmic Reticulum-to-Golgi Transport but Does Not Promote SNARE Motif Accessibility

2004 ◽  
Vol 15 (1) ◽  
pp. 162-175 ◽  
Author(s):  
Antionette L. Williams ◽  
Sebastian Ehm ◽  
Noëlle C. Jacobson ◽  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Protein Function ◽  
Snare Complex ◽  
Golgi Transport ◽  
Snare Motif ◽  
Sm Protein ◽  
Snare Complex Formation

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE–complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1–syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.

scholarly journals Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

1990 ◽  
Vol 10 (5) ◽  
pp. 1989-2001 ◽  
Author(s):  
D T Ng ◽  
S W Hiebert ◽  
R A Lamb
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Carbohydrate Chain ◽  
Protein A ◽  
Simian Virus ◽  
Glycosylation Pattern ◽  
Transient Complex ◽  
Simian Virus 5 ◽  
Endoplasmic Reticulum Protein ◽  
Carbohydrate Chains

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.

scholarly journals Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

1988 ◽  
Vol 8 (9) ◽  
pp. 3740-3747 ◽  
Author(s):  
H W Stürzbecher ◽  
C Addison ◽  
J R Jenkins
Keyword(s):  
Complex Formation ◽  
Protein Complexes ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mutant P53 ◽  
Cos Cells

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

scholarly journals A Cdc48 “Retrochaperone” Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates

2017 ◽  
Vol 292 (8) ◽  
pp. 3112-3128 ◽  
Author(s):  
Sonya Neal ◽  
Raymond Mak ◽  
Eric J. Bennett ◽  
Randolph Hampton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Full Length ◽  
26S Proteasome ◽  
Polyubiquitin Chains ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Erad Pathway ◽  
Er Membrane

A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical “retrochaperone” for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.

scholarly journals Glycosylation Can Influence Topogenesis of Membrane Proteins and Reveals Dynamic Reorientation of Nascent Polypeptides within the Translocon

1999 ◽  
Vol 147 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Veit Goder ◽  
Christoph Bieri ◽  
Martin Spiess
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Dynamic Process ◽  
Membrane Targeting ◽  
Protein Orientation ◽  
Nascent Polypeptide ◽  
Targeting Efficiency ◽  
Internal Signal ◽  
Chimeric Model

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH2-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH2-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.

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