scholarly journals Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment.

1995 ◽  
Vol 130 (1) ◽  
pp. 1-13 ◽  
Author(s):  
F Korioth ◽  
C Gieffers ◽  
G G Maul ◽  
J Frey
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Coiled Coil ◽  
Nuclear Staining ◽  
Interferon Treatment ◽  
Immunofluorescence Analysis ◽  
Host Interactions ◽  
Nuclear Domain ◽  
Novel Protein

The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus-host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle.

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

scholarly journals A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

1997 ◽  
Vol 17 (11) ◽  
pp. 6683-6692 ◽  
Author(s):  
A J Peterson ◽  
M Kyba ◽  
D Bornemann ◽  
K Morgan ◽  
H W Brock ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Direct Interaction ◽  
Molecular Complexes ◽  
Polycomb Group ◽  
Ph Domain ◽  
Binding Studies

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

scholarly journals Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

1999 ◽  
Vol 147 (7) ◽  
pp. 1569-1582 ◽  
Author(s):  
Michelangelo Cordenonsi ◽  
Fabio D'Atri ◽  
Eva Hammar ◽  
David A.D. Parry ◽  
John Kendrick-Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Coiled Coil ◽  
Full Length ◽  
Cytoplasmic Surface ◽  
Terminal Fragment ◽  
Reticulocyte Lysates ◽  
Actomyosin Cytoskeleton

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

scholarly journals Proximity Labeling To Map Host-Pathogen Interactions at the Membrane of a Bacterium-Containing Vacuole in Chlamydia trachomatis-Infected Human Cells

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Macy G. Olson ◽  
Ray E. Widner ◽  
Lisa M. Jorgenson ◽  
Alyssa Lawrence ◽  
Dragana Lagundzin ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Inclusion Membrane ◽  
Content Type ◽  
Host Interactions ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Chlamydial Inclusion

ABSTRACT Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.

scholarly journals Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

2002 ◽  
Vol 22 (15) ◽  
pp. 5451-5466 ◽  
Author(s):  
Suzanne E. Wardell ◽  
Viroj Boonyaratanakornkit ◽  
James S. Adelman ◽  
Ami Aronheim ◽  
Dean P. Edwards
Keyword(s):  
Progesterone Receptor ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Physiological Role ◽  
Target Cells ◽  
Basic Leucine Zipper ◽  
Interacting Protein

ABSTRACT The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that functions as an AP-1 repressor. PR and JDP-2 interact directly in vitro through the DNA binding domain (DBD) of PR and the basic leucine zipper (bZIP) region of JDP-2. The two proteins also physically associate in mammalian cells, as detected by coimmunoprecipitation, and are recruited in vivo to a progesterone-inducible target gene promoter, as detected by a chromatin immunoprecipitation (ChIP) assay. In cell transfection assays, JDP-2 substantially increased hormone-dependent PR-mediated transactivation and worked primarily by stimulating AF-1 activity. JDP-2 is a substantially stronger coactivator of AF-1 than SRC-1 and stimulates AF-1 independent of SRC-1 pathways. The PR DBD is necessary but not sufficient for JDP-2 stimulation of PR activity; the DBD and AF-1 are required together. JDP-2 lacks an intrinsic activation domain and makes direct protein interactions with other coactivators, including CBP and p300 CBP-associated factor (pCAF), but not with SRCs. These results indicate that JDP-2 stimulates AF-1 activity by the novel mechanism of docking to the DBD and recruiting or stabilizing N-terminal PR interactions with other general coactivators. JDP-2 has preferential activity on PR among the nuclear receptors tested and is expressed in progesterone target cells and tissues, suggesting that it has a physiological role in PR function.

scholarly journals Redox-dependent oligomerization through a leucine zipper motif is essential for MG53-mediated cell membrane repair

2011 ◽  
Vol 301 (1) ◽  
pp. C106-C114 ◽  
Author(s):  
Moonsun Hwang ◽  
Jae-kyun Ko ◽  
Noah Weisleder ◽  
Hiroshi Takeshima ◽  
Jianjie Ma
Keyword(s):  
Cell Membrane ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Mechanical Damage ◽  
Thiol Group ◽  
Membrane Repair ◽  
Coiled Coil Domain ◽  
Green Fluorescent

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

scholarly journals Protein–Protein Interactions Governing Septin Heteropentamer Assembly and Septin Filament Organization in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (10) ◽  
pp. 4568-4583 ◽  
Author(s):  
Matthias Versele ◽  
Björn Gullbrand ◽  
Mark J. Shulewitz ◽  
Victor J. Cid ◽  
Shirin Bahmanyar ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Coiled Coil ◽  
Microscopic Analysis ◽  
Protein Protein Interactions ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Necessary And Sufficient ◽  
Septin Filament

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3–Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

scholarly journals Human Cytomegalovirus UL84 Oligomerization and Heterodimerization Domains Act as Transdominant Inhibitors of oriLyt-Dependent DNA Replication: Evidence that IE2-UL84 and UL84-UL84 Interactions Are Required for Lytic DNA Replication

2004 ◽  
Vol 78 (17) ◽  
pp. 9203-9214 ◽  
Author(s):  
Kelly S. Colletti ◽  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Margaret Tarrant ◽  
Gregory S. Pari
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Protein Interactions ◽  
Leucine Zipper ◽  
Lytic Replication ◽  
Protein Domain ◽  
Protein Protein Interactions

ABSTRACT Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.

scholarly journals Revelation of a Novel Protein Translocon in Bacterial Plasma Membrane

2017 ◽  
Author(s):  
Feng Jin ◽  
Zengyi Chang
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Living Cell ◽  
Outer Membrane Proteins ◽  
Bacterial Cells ◽  
Bam Complex ◽  
Gxxxg Motif ◽  
Novel Protein

Many proteins are translocated across biomembranes via protein translocons in targeting to their subcellular destinations. Hitherto, the SecYEG/Sec61 translocon, existing in prokaryotes and eukaryotes, represents the most intensively studied one. According to the current perception, both periplasmic and β-barrel outer membrane proteins (β-barrel OMPs) are translocated via the SecYEG translocon in bacterial cells, although direct living cell evidences remain lacking. Here, mainly viain vivoprotein photo-crosslinking analysis, we revealed that the never reported membrane-integrated SecANprotein apparently functions as the translocon for β-barrel OMPs. Additionally, SecANcontains a GXXXG motif known for mediating protein interactions in biomembranes, and processing of β-barrel OMP precursors was severely affected in cells producing an assembly-defective SecANvariant resulted from the GXXXG motif mutations. Furthermore, SecANwas demonstrated to directly interact with the Bam complex, thus likely be a part of the supercomplex that we revealed earlier to be responsible for β-barrel OMP biogenesis.

scholarly journals The golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs

2017 ◽  
Vol 28 (20) ◽  
pp. 2686-2700 ◽  
Author(s):  
Nadine S. Anderson ◽  
Indrani Mukherjee ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Retrograde Transport ◽  
Snare Proteins ◽  
Growth Defects ◽  
Cog Complex ◽  
Physical Interactions ◽  
And Function

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

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