scholarly journals Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor

2003 ◽  
Vol 372 (2) ◽  
pp. 453-463 ◽  
Author(s):  
Marianne TARDIF ◽  
Laurence BROUCHON ◽  
Marie-Josèphe RABIET ◽  
François BOULAY
Keyword(s):  
Active Form ◽  
Hybrid Approach ◽  
Full Length ◽  
Actin Dynamics ◽  
Yeast Two Hybrid ◽  
C5a Receptor ◽  
Wiskott Aldrich Syndrome ◽  
C5a Anaphylatoxin ◽  
Syndrome Protein ◽  
Two Hybrid

Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott–Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1–321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active ‘open conformation’, the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals A novel yeast two-hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein1

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 904-911 ◽  
Author(s):  
Miguel A. Rodriguez Milla ◽  
Yuichi Uno ◽  
Ing-Feng Chang ◽  
Jared Townsend ◽  
Eileen A. Maher ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Hybrid Approach ◽  
Yeast Two Hybrid ◽  
Two Hybrid

A Method to Generate Full-Length cDNA Molecules from Yeast Two-Hybrid Clones and RACE Products

1999 ◽  
Vol 268 (1) ◽  
pp. 161-162 ◽  
Author(s):  
Charles S. Hemenway ◽  
Benjamin W. Halligan ◽  
Grahame C.D. Gould
Keyword(s):  
Full Length ◽  
Yeast Two Hybrid ◽  
Full Length Cdna ◽  
Hybrid Clones ◽  
Two Hybrid

Protein interactions of fatty acid synthase II

2000 ◽  
Vol 28 (6) ◽  
pp. 615-616 ◽  
Author(s):  
G. Honeyman ◽  
T. Fawcett
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Hybrid Approach ◽  
Carrier Protein ◽  
Yeast Two Hybrid ◽  
Two Hybrid

We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components. Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus.

scholarly journals SERTA Domain Containing Protein 1 (SERTAD1) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2, Which Is Involved in Virus Virulence in Swine

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 421
Author(s):  
Elizabeth A. Vuono ◽  
Elizabeth Ramirez-Medina ◽  
Paul Azzinaro ◽  
Keith A. Berggren ◽  
Ayushi Rai ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Classical Swine Fever Virus ◽  
Critical Role ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Proximity Ligation Assay ◽  
Yeast Two Hybrid ◽  
Clinically Normal ◽  
Two Hybrid

E2 is the major structural glycoprotein of the classical swine fever virus (CSFV). E2 has been shown to be involved in important virus functions such as replication and virulence in swine. Using the yeast two-hybrid system, we previously identified several host proteins specifically interacting with CSFV E2. Here, we analyze the protein interaction of E2 with SERTA domain containing protein 1 (SERTAD1), a factor involved in the stimulation of the transcriptional activities of different host genes. We have confirmed that the interaction between these two proteins occurs in CSFV-infected swine cells by using a proximity ligation assay and confocal microscopy. Amino acid residues in the CSFV E2 protein that are responsible for mediating the interaction with SERTAD1 were mapped by a yeast two-hybrid approach using a randomly mutated E2 library. Using that information, a recombinant CSFV mutant (E2ΔSERTAD1v) that harbors substitutions in those residues mediating the protein-interaction with SERTAD1 was developed and used to study the role of the E2-SERTAD1 interaction in viral replication and virulence in swine. CSFV E2ΔSERTAD1v, when compared to the parental BICv, showed a clearly decreased ability to replicate in the SK6 swine cell line and a more severe replication defect in primary swine macrophage cultures. Importantly, 80% of animals infected with E2ΔSERTAD1v survived infection, remaining clinically normal during the 21-day observational period. This result would indicate that the ability of CSFV E2 to bind host SERTAD1 protein during infection plays a critical role in virus virulence.

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