Faculty Opinions recommendation of Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus.

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

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The Drosophila pipsqueak gene encodes a nuclear BTB-domain-containing protein required early in oogenesis

Development ◽

10.1242/dev.122.6.1859 ◽

1996 ◽

Vol 122 (6) ◽

pp. 1859-1871 ◽

Cited By ~ 1

Author(s):

H. Horowitz ◽

C.A. Berg

Keyword(s):

Fusion Protein ◽

Protein Interactions ◽

Protein Isoforms ◽

Follicle Cells ◽

Protein Protein Interactions ◽

Gene Encoding ◽

Btb Domain ◽

Multiple Transcripts ◽

C Terminus ◽

Evolutionarily Conserved

Mutations at the pipsqueak locus affect early patterning in the Drosophila egg and embryo. We have cloned pipsqueak and found that it is a large and complex gene, encoding multiple transcripts and protein isoforms. One protein, PsqA, is absent in all of the mutants that we have examined. We show that PsqA is a nuclear protein present in the germ cells and somatically derived follicle cells throughout oogenesis and that it is required prior to stage one of oogenesis. PsqA contains a BTB (POZ) domain at its amino terminus; additionally, we have identified an evolutionarily conserved motif of unknown function present four times in tandem at the C terminus of the protein. PZ pipsqueak mutants produce a putative fusion protein containing the pipsqueak BTB domain fused to sequences resident on the PZ element (H. Horowitz and C. Berg, 1995 Genetics 139, 327–335). We demonstrate here that expression of this fusion protein in wild-type flies has a dominant effect, resulting in infertility and eggshell defects. These dominant phenotypes are discussed in light of current theories on the role of the BTB domain in protein-protein interactions.

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Determining Protein−Protein Interactions by Oxidative Cross-Linking of a Glycine-Glycine-Histidine Fusion Protein†

Biochemistry ◽

10.1021/bi9728046 ◽

1998 ◽

Vol 37 (13) ◽

pp. 4397-4406 ◽

Cited By ~ 27

Author(s):

Kathlynn C. Brown ◽

Zhonghua Yu ◽

Alma L. Burlingame ◽

Charles S. Craik

Keyword(s):

Fusion Protein ◽

Protein Interactions ◽

Cross Linking ◽

Protein Protein Interactions

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Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

Molecular Biology of the Cell ◽

10.1091/mbc.7.5.693 ◽

1996 ◽

Vol 7 (5) ◽

pp. 693-701 ◽

Cited By ~ 40

Author(s):

R J Barnard ◽

A Morgan ◽

R D Burgoyne

Keyword(s):

Membrane Proteins ◽

Fusion Protein ◽

Conformational Changes ◽

Protein Interactions ◽

Atp Hydrolysis ◽

Coiled Coil ◽

Protein Protein Interactions ◽

Permeabilized Cells ◽

C Terminus ◽

Adrenal Chromaffin

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.

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Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

10.1101/2020.09.23.309088 ◽

2020 ◽

Author(s):

Zhiqiang Fu ◽

Yasmine Rais ◽

X. Chris Le ◽

Andrei P. Drabovich

Keyword(s):

Prostate Cancer ◽

Mass Spectrometry ◽

Fusion Protein ◽

Protein Interactions ◽

Transcript Level ◽

Protein Isoforms ◽

Protein Protein Interactions ◽

Molecular Alteration ◽

And Function ◽

First Time

SummaryTMPRSS2-ERG gene fusion, a molecular alteration driving nearly a half of prostate cancer cases, has been intensively characterized at the transcript level, while limited studies explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed and applied immunoprecipitation-mass spectrometry (IP-MS) assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells. IP-MS assays quantified total ERG (∼27,000 copies/cell) and its four unique isoforms, and revealed that the T1E4-ERG isoform accounts for 71% of the total ERG protein in VCaP cells. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified and quantified. IP-MS with the C-terminal antibodies identified 29 proteins in the ERG interactome, including SWI/SNF chromatin remodeling complex subunits and numerous transcriptional co-regulators. Our data also suggested that TMPRSS2-ERG protein-protein interactions were exerted through at least two different regions. Knowledge on the distinct TMPRSS2-ERG protein isoforms and interactomes may facilitate development of more accurate diagnostics and targeted therapeutics of prostate cancer.

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Biomolecular condensates amplify mRNA decapping by coupling protein interactions with conformational changes in Dcp1/Dcp2

10.1101/2020.07.09.195057 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ryan W. Tibble ◽

Anaïs Depaix ◽

Joanna Kowalska ◽

Jacek Jemielity ◽

John D. Gross

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Protein Interactions ◽

Mrna Degradation ◽

Enzyme Activation ◽

List Type ◽

Protein Protein Interactions ◽

Mrna Decapping ◽

P Bodies

SUMMARYCells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates enriched in factors important for mRNA degradation. P-bodies have been identified as sites of both mRNA storage and decay, but how these opposing outcomes may be achieved in condensates is unresolved. A critical step in mRNA degradation is removal of the 5’-7-methylguanosine cap by Dcp1/Dcp2, which is highly enriched in P-bodies. Dcp1/Dcp2 activity is repressed in condensates in vitro and requires the activator Edc3. Activation of decapping is amplified in condensates relative to the surrounding solution due to stabilization of an autoinhibited state in Dcp1/Dcp2. Edc3 couples a conformational change in the Dcp1/Dcp2 active site with alteration of the protein-protein interactions driving phase separation to activate decapping in condensates. The composition-dependent regulation of enzyme activity in condensates occurs over length scales ranging from microns to Ångstroms and may control the functional state of P-bodies and related phase-separated compartments.HIGHLIGHTSmRNA decapping in droplets is repressedCatalytically inert droplets are activated by a change in condensate compositionA switch in enzymatic activity requires a conformational change in condensatesCondensates amplify enzyme activation compared to surrounding solution

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