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

Blood ◽  
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.

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

2020 ◽  
Vol 21 (9) ◽  
pp. 3193 ◽  
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.

A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1552-1552
Author(s):  
Hirohiko Shibayama ◽  
Yuri Saito ◽  
Akira Tanimura ◽  
Hirokazu Tanaka ◽  
Itaru Matsumura ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Cyclin D1 ◽  
P38 Mapk ◽  
Conflicts Of Interest ◽  
Biological Effects ◽  
Terminal Region ◽  
Yeast Two Hybrid ◽  
A Cell ◽  
Two Hybrid

Abstract Abstract 1552 [Background] Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated. [Methods & Results] In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells. [Conclusion] P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals γ-Synergin

1999 ◽  
Vol 146 (5) ◽  
pp. 993-1004 ◽  
Author(s):  
Lesley J. Page ◽  
Penelope J. Sowerby ◽  
Winnie W.Y. Lui ◽  
Margaret S. Robinson
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Yeast Two Hybrid ◽  
Eh Domain ◽  
Library Screen ◽  
Alternatively Spliced ◽  
Vesicle Cycle ◽  
Two Hybrid

The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the γ-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both γ-adaptin and α-adaptin, and γ-synergin, an alternatively spliced protein with an apparent molecular mass of ∼110–190 kD, which only interacts with γ-adaptin. γ-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of γ-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the γ-adaptin binding site. In cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, much of the γ-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that γ-synergin links the AP-1 complex to another protein or proteins.

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

2009 ◽  
Vol 14 (2) ◽  
Author(s):  
Haifeng Weng ◽  
Chul Kim ◽  
Christos Valavanis ◽  
Zhaohui Wang ◽  
Lawrence Schwartz
Keyword(s):  
Transcription Factors ◽  
Family Member ◽  
Cdna Library ◽  
Mouse Embryo ◽  
Yeast Two Hybrid ◽  
Binding Partner ◽  
C Terminus ◽  
La Antigen ◽  
Two Hybrid

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.

scholarly journals The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis

Blood ◽  
2012 ◽  
Vol 120 (25) ◽  
pp. 5032-5040 ◽  
Author(s):  
Denisa Urban ◽  
Ling Li ◽  
Hilary Christensen ◽  
Fred G. Pluthero ◽  
Shao Zun Chen ◽  
...  
Keyword(s):  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Yeast Two Hybrid ◽  
Binding Partner ◽  
Late Endosomes ◽  
Arc Syndrome ◽  
Membrane Bound ◽  
Golgi Network ◽  
Two Hybrid

Abstract Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

scholarly journals Murine Fam65b forms ring-like structures at the base of stereocilia critical for mechanosensory hair cell function

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Bo Zhao ◽  
Zizhen Wu ◽  
Ulrich Müller
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Cell Function ◽  
Yeast Two Hybrid ◽  
Cochlear Hair Cells ◽  
Unknown Mechanism ◽  
Binding Partner ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction ◽  
Two Hybrid

Cochlear hair cells convert sound-induced vibration into electrical signals. FAM65B mutations cause hearing loss by an unknown mechanism. Using biochemistry and stochastic optical reconstruction microscopy (STORM), we show here that Fam65b oligomers form a circumferential ring near the basal taper of the mechanically sensitive stereocilia of murine hair cells. Taperin, a second protein near the taper, forms a dense-core-like structure that is disrupted in the absence of Fam65b. Stereocilia of Fam65b-deficient murine hair cells start to develop, but mechanotransduction is affected and stereocilia deteriorate. Yeast-two-hybrid screens identify RhoC as a Fam65b binding partner. RhoC co-localizes with Fam65b in stereocilia and regulates Fam65b oligomerization. Binding to RhoC and oligomerization are critical for Fam65b function. Our findings thus reveal a highly organized compartment near the base of stereocilia that is critical for hair cell function and affected in disease.

scholarly journals Saccharomyces cerevisiae Rbg1 Protein and Its Binding Partner Gir2 Interact on Polyribosomes with Gcn1

2009 ◽  
Vol 8 (7) ◽  
pp. 1061-1071 ◽  
Author(s):  
P. K. Wout ◽  
E. Sattlegger ◽  
S. M. Sullivan ◽  
J. R. Maddock
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Yeast Two Hybrid ◽  
Uncharacterized Protein ◽  
General Amino Acid Control ◽  
Binding Partner ◽  
Inhibition Of Growth ◽  
Fractionation Pattern ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT Rbg1 is a previously uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg/CgtA subfamily of GTP-binding proteins whose members are involved in ribosome function in both prokaryotes and eukaryotes. We show here that Rbg1 specifically associates with translating ribosomes. In addition, in this study proteins were identified that interact with Rbg1 by yeast two-hybrid screening and include Tma46, Ygr250c, Yap1, and Gir2. Gir2 contains a GI (Gcn2 and Impact) domain similar to that of Gcn2, an essential factor of the general amino acid control pathway required for overcoming amino acid shortage. Interestingly, we found that Gir2, like Gcn2, interacts with Gcn1 through its GI domain, and overexpression of Gir2, under conditions mimicking amino acid starvation, resulted in inhibition of growth that could be reversed by Gcn2 co-overexpression. Moreover, we found that Gir2 also cofractionated with polyribosomes, and this fractionation pattern was partially dependent on the presence of Gcn1. Based on these findings, we conclude that Rbg1 and its interacting partner Gir2 associate with ribosomes, and their possible biological roles are discussed.

scholarly journals Construction of a reading frame–independent yeast two-hybrid vector system for site-specific recombinational cloning and protein interaction screening

BioTechniques ◽  
2008 ◽  
Vol 45 (3) ◽  
pp. 235-244 ◽  
Author(s):  
Richard H. Maier ◽  
Christina J. Brandner ◽  
Helmut Hintner ◽  
Johann W. Bauer ◽  
Kamil Önder
Keyword(s):  
Protein Interaction ◽  
Vector System ◽  
Yeast Two Hybrid ◽  
Site Specific ◽  
Reading Frame ◽  
Interaction Screening ◽  
Two Hybrid

scholarly journals Murine CENP-F Regulates Centrosomal Microtubule Nucleation and Interacts with Hook2 at the Centrosome

2009 ◽  
Vol 20 (22) ◽  
pp. 4790-4803 ◽  
Author(s):  
Katherine L. Moynihan ◽  
Ryan Pooley ◽  
Paul M. Miller ◽  
Irina Kaverina ◽  
David M. Bader
Keyword(s):  
Protein Function ◽  
Structure And Function ◽  
The Novel ◽  
Yeast Two Hybrid ◽  
Cellular Functions ◽  
Binding Partner ◽  
Broad Array ◽  
Organizing Center ◽  
And Function ◽  
Two Hybrid

The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F−/− cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F−/− cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.

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