Acheron, an novel LA antigen family member, binds to cask and forms a complex with id transcription factors

AbstractAcheron, a Lupus antigen ortholog, was identified as a novel death-associated transcript from the intersegmental muscles of the mothManduca sexta. Acheron is phylogenetically-conserved and represents a new sub-family of Lupus antigen proteins. Acheron is expressed predominantly in neurons and muscle in vertebrates, and regulates several developmental events including myogenesis, neurogenesis and possibly metastasis. Using Acheron as bait, we performed a yeast two-hybrid screen with a mouse embryo cDNA library and identified CASK-C, a novel CASK/Lin-2 isoform, as an Acheron binding partner. Acheron and CASK-C bind via the C-terminus of Acheron and the CaMKII-like domain of CASK-C. Co-immunoprecipitation assays verify this interaction and demonstrate that Acheron also forms a complex with all members of the Id (inhibitor of differentiation) proteins. Taken together, these data suggest a mechanism by which Acheron may regulate development and pathology.

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The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽

10.1099/mic.0.26997-0 ◽

2004 ◽

Vol 150 (7) ◽

pp. 2055-2068 ◽

Cited By ~ 10

Author(s):

Daniel V. Zurawski ◽

Murry A. Stein

Keyword(s):

Type Iii Secretion ◽

Coiled Coil ◽

Amphipathic Helix ◽

Yeast Two Hybrid ◽

C Terminus ◽

N Terminus ◽

Domain Mapping ◽

Self Association ◽

Discrete Domains ◽

Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

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Screening of binding proteins that interact with human Salvador 1 in a human fetal liver cDNA library by the yeast two-hybrid system

Molecular Biology Reports ◽

10.1007/s11033-012-1670-4 ◽

2012 ◽

Vol 39 (8) ◽

pp. 8225-8230 ◽

Cited By ~ 4

Author(s):

Xiaolan Li ◽

Xuelai Luo ◽

Zhaoming Li ◽

Guihua Wang ◽

Hui Xiao ◽

...

Keyword(s):

Cdna Library ◽

Hybrid System ◽

Binding Proteins ◽

Fetal Liver ◽

Yeast Two Hybrid ◽

Human Fetal Liver ◽

Yeast Two Hybrid System ◽

Liver Cdna Library ◽

Two Hybrid

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Identification of the multivalent PDZ domain protein PDZK1 as a binding partner of sodium‐coupled monocarboxylate cotransporter 2 (SMCT2) by yeast two‐hybrid assay

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.1204.3 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Sunena Srivastava ◽

Naohiko Anzai ◽

Ai Yamanishi ◽

Vadivel Ganapathy ◽

Hitoshi Endou

Keyword(s):

Pdz Domain ◽

Yeast Two Hybrid ◽

Binding Partner ◽

Domain Protein ◽

Two Hybrid

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Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

Journal of Biological Chemistry ◽

10.1074/jbc.m009810200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11980-11987 ◽

Cited By ~ 88

Author(s):

Steven A. Haney ◽

Elizabeth Glasfeld ◽

Cynthia Hale ◽

David Keeney ◽

Zhizhen He ◽

...

Keyword(s):

Hybrid System ◽

Enzyme Linked Immunosorbent Assay ◽

Wild Type ◽

Yeast Two Hybrid ◽

Conserved Sequence ◽

Yeast Two Hybrid System ◽

C Terminus ◽

Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

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Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

International Journal of Molecular Sciences ◽

10.3390/ijms21093193 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3193 ◽

Cited By ~ 1

Author(s):

Juan Luis Araujo-Garrido ◽

Fernando Baisón-Olmo ◽

Joaquín Bernal-Bayard ◽

Francisco Romero ◽

Francisco Ramos-Morales

Keyword(s):

Host Cell ◽

Type Iii Secretion ◽

Salmonella Enterica Serovar Typhimurium ◽

Yeast Two Hybrid ◽

Microtubule Network ◽

Binding Partner ◽

Tubulin Binding ◽

Serovar Typhimurium ◽

Novel Target ◽

Two Hybrid

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

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Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.

Blood ◽

10.1182/blood.v116.21.1444.1444 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1444-1444

Author(s):

Denisa Urban ◽

Ling Li ◽

James Wasmuth ◽

Hilary Christensen ◽

John Parkinson ◽

...

Keyword(s):

Conflicts Of Interest ◽

Human Platelets ◽

Multiprotein Complex ◽

Yeast Two Hybrid ◽

Granule Formation ◽

Reading Frame ◽

Binding Partner ◽

Library Screen ◽

Two Hybrid ◽

Granule Release

Abstract Abstract 1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis. Disclosures: No relevant conflicts of interest to declare.

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OL-049 Screening of proteins binding to HCV NS3 protein from human pancreas cDNA library by yeast two-hybrid

International Journal of Infectious Diseases ◽

10.1016/s1201-9712(09)60146-6 ◽

2008 ◽

Vol 12 ◽

pp. S52

Author(s):

C.C. Hu ◽

J.Q. Zhang ◽

X.C. Wang ◽

G.L. Li ◽

Q. Wang ◽

...

Keyword(s):

Cdna Library ◽

Human Pancreas ◽

Yeast Two Hybrid ◽

Ns3 Protein ◽

Two Hybrid

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Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo

Development ◽

10.1242/dev.125.16.3111 ◽

1998 ◽

Vol 125 (16) ◽

pp. 3111-3121 ◽

Cited By ~ 16

Author(s):

M. Sefton ◽

S. Sanchez ◽

M.A. Nieto

Keyword(s):

Transcription Factors ◽

Amino Acid ◽

Chick Embryo ◽

Neural Crest ◽

Family Member ◽

Zinc Finger ◽

Mouse Embryo ◽

Amino Acid Sequences ◽

Mouse Homologue ◽

Snail Family

The members of the Snail family of zinc-finger transcription factors have been implicated in the formation of distinct tissues within the developing vertebrate and invertebrate embryo. Two members of this family have been described in higher vertebrates, Snail (Sna) and Slug (Slu), where they have been implicated in the formation of tissues such as the mesoderm and the neural crest. We have isolated the mouse homologue of the Slu gene enabling us to analyse and compare the amino acid sequences and the patterns of expression of both Sna and Slu in the chick and mouse. We have detected features in the sequences that allow the unequivocal ascription of any family member to the Sna or Slu subfamilies and we have observed that, during early stages of development, many of the sites of Slu and Sna expression in the mouse and chick embryo are swapped. Later in development, the sites of expression of Slu and Sna are conserved between these two species. These data, together with the data available in other species, lead us to propose that Slu and Sna arose as a duplication of an ancestor gene and that an extra duplication in the fish lineage has given rise to two Sna genes. Furthermore, several early sites of Slu and Sna expression have been swapped in the avian lineage. Our analysis of the Snail family may also shed new light on the origin of the neural crest.

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