scholarly journals Characterisation of class VI TRIM RING domains: linking RING activity to C-terminal domain identity

2019 ◽  
Vol 2 (3) ◽  
pp. e201900295 ◽  
Author(s):  
Rebecca V Stevens ◽  
Diego Esposito ◽  
Katrin Rittinger
Keyword(s):  
Solution Structure ◽  
Coiled Coil ◽  
E3 Ligase ◽  
Cellular Processes ◽  
Terminal Domain ◽  
Coiled Coil Region ◽  
Biochemical Characterisation ◽  
Ring Domains ◽  
Self Association ◽  
Terminal Domains

TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.

scholarly journals Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases

2020 ◽  
Vol 48 (6) ◽  
pp. 2615-2624
Author(s):  
Filippo Fiorentini ◽  
Diego Esposito ◽  
Katrin Rittinger
Keyword(s):  
Coiled Coil ◽  
E3 Ligase ◽  
Protein Family ◽  
Ring Domain ◽  
Cellular Functions ◽  
Protein Activity ◽  
Coiled Coil Region ◽  
Tripartite Motif ◽  
Large Protein Family ◽  
Self Association

TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.

scholarly journals SVIP Is a Novel VCP/p97-interacting Protein Whose Expression Causes Cell Vacuolation

2003 ◽  
Vol 14 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Masami Nagahama ◽  
Mie Suzuki ◽  
Yuko Hamada ◽  
Kiyotaka Hatsuzawa ◽  
Katsuko Tani ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coiled Coil ◽  
Adaptor Proteins ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Acid Protein ◽  
Extensive Cell ◽  
Dependent Protein ◽  
Amino Acid Protein

VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation. It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97. To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named smallVCP/p97-interactingprotein (SVIP). Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus. Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other. SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner. Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells. The vacuoles seemed to be derived from endoplasmic reticulum membranes. These results together suggest that SVIP is a novelVCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum.

scholarly journals Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions

2007 ◽  
Vol 189 (24) ◽  
pp. 8801-8806 ◽  
Author(s):  
Claire Verbelen ◽  
Dominique Raze ◽  
Frédérique Dewitte ◽  
Camille Locht ◽  
Yves F. Dufrêne
Keyword(s):  
Single Molecule ◽  
Coiled Coil ◽  
Bimodal Distribution ◽  
Heparin Binding ◽  
Force Microscopy ◽  
Bacterial Aggregation ◽  
Atomic Force ◽  
Coiled Coil Region ◽  
Force Curves ◽  
Terminal Domains

ABSTRACT The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.

scholarly journals Megator, an Essential Coiled-Coil Protein that Localizes to the Putative Spindle Matrix during Mitosis inDrosophila

2004 ◽  
Vol 15 (11) ◽  
pp. 4854-4865 ◽  
Author(s):  
Hongying Qi ◽  
Uttama Rath ◽  
Dong Wang ◽  
Ying-Zhi Xu ◽  
Yun Ding ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Matrix Proteins ◽  
P Element ◽  
Structural Basis ◽  
Spindle Matrix ◽  
Terminal Domain ◽  
Coiled Coil Domain ◽  
Coiled Coil Region ◽  
Spherical Structures ◽  
Spindle Structure

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

scholarly journals The E3 Ligase CHIP: Insights into Its Structure and Regulation

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Indranil Paul ◽  
Mrinal K. Ghosh
Keyword(s):  
Quality Control ◽  
Tandem Repeats ◽  
Current Knowledge ◽  
Coiled Coil ◽  
E3 Ligase ◽  
Review Article ◽  
Regulatory Mechanisms ◽  
Carboxy Terminus ◽  
Interacting Protein ◽  
Coiled Coil Region

The carboxy-terminus of Hsc70 interacting protein (CHIP) is a cochaperone E3 ligase containing three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region. CHIP is known to function as a central quality control E3 ligase and regulates several proteins involved in a myriad of physiological and pathological processes. Recent studies have highlighted varied regulatory mechanisms operating on the activity of CHIP which is crucial for cellular homeostasis. In this review article, we give a concise account of our current knowledge on the biochemistry and regulation of CHIP.

scholarly journals Genetic Analysis of hook, a Gene Required for Endocytic Trafficking in Drosophila

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 675-684
Author(s):  
Helmut Krämer ◽  
Meridee Phistry
Keyword(s):  
Genetic Analysis ◽  
Coiled Coil ◽  
Complete Loss ◽  
Endocytic Trafficking ◽  
Multivesicular Bodies ◽  
Loss Of Function ◽  
Endocytic Compartment ◽  
Coiled Coil Region ◽  
Null Phenotype ◽  
Terminal Domains

Abstract The Drosophila hook gene encodes a novel component of the endocytic compartment. Previously identified hook alleles, which still expressed truncated Hook proteins, affected the accumulation of internalized transmembrane ligands into multivesicular bodies (MVBs). To determine the hook null phenotype, we isolated nine new hook alleles on the basis of their characteristic hooked-bristle phenotype. At least one of these alleles, hk11, is a complete loss-of-function allele. Flies carrying the hk11 allele are viable and fertile but neither transmembrane ligands nor soluble ligands accumulate in MVBs. This effect on endocytosed ligands can be mimicked by the expression of Hook proteins truncated for the N- and C-terminal domains flanking the central coiled-coil region. The importance of all three domains for Hook function was confirmed by their conservation between two Drosophila and two human Hook proteins.

Functional interactions between ubiquitin E2 enzymes and TRIM proteins

2011 ◽  
Vol 434 (2) ◽  
pp. 309-319 ◽  
Author(s):  
Luisa M. Napolitano ◽  
Ellis G. Jaffray ◽  
Ronald T. Hay ◽  
Germana Meroni
Keyword(s):  
Coiled Coil ◽  
Specific Interactions ◽  
E3 Ligases ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Physiological Relevance ◽  
Tripartite Motif ◽  
E2 Enzymes ◽  
Specific Reactions ◽  
Trim Proteins

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM–UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.

scholarly journals Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

2008 ◽  
Vol 8 (2) ◽  
pp. 134-146 ◽  
Author(s):  
Bhaskarjyoti Sarmah ◽  
Susan R. Wente
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Morphology ◽  
Mrna Export ◽  
Coiled Coil ◽  
Polarized Growth ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Cellular Processes ◽  
Terminal Domain ◽  
Carboxy Terminal

ABSTRACT The inositol 1,3,4,5,6-pentakisphosphate (IP5) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP6) in eukaryotic cells. Previous studies have shown that IP6 is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1 + in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1 + deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP6 production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1 + function, encoding an IP6 kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1 + upstream of ipk1 +. We propose that IP6 production is a key signaling linchpin for regulating multiple essential cellular processes.

scholarly journals Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

2012 ◽  
Vol 199 (1) ◽  
pp. 111-124 ◽  
Author(s):  
Qing-Tao Shen ◽  
Peter P. Hsiue ◽  
Charles V. Sindelar ◽  
Matthew D. Welch ◽  
Kenneth G. Campellone ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Coiled Coil ◽  
Tubular Structures ◽  
Terminal Portion ◽  
Actin Nucleation ◽  
Cellular Processes ◽  
Coiled Coil Region ◽  
Membrane Tubulation ◽  
Underlying Mechanisms ◽  
Structural Insights

The microtubule (MT) and actin cytoskeletons drive many essential cellular processes, yet fairly little is known about how their functions are coordinated. One factor that mediates important cross talk between these two systems is WHAMM, a Golgi-associated protein that utilizes MT binding and actin nucleation activities to promote membrane tubulation during intracellular transport. Using cryoelectron microscopy and other biophysical and biochemical approaches, we unveil the underlying mechanisms for how these activities are coordinated. We find that WHAMM bound to the outer surface of MT protofilaments via a novel interaction between its central coiled-coil region and tubulin heterodimers. Upon the assembly of WHAMM onto MTs, its N-terminal membrane-binding domain was exposed at the MT periphery, where it can recruit vesicles and remodel them into tubular structures. In contrast, MT binding masked the C-terminal portion of WHAMM and prevented it from promoting actin nucleation. These results give rise to a model whereby distinct MT-bound and actin-nucleating populations of WHAMM collaborate during membrane tubulation.

scholarly journals Relevance of Ebola virus VP35 homo-dimerization on the type I interferon cascade inhibition

2019 ◽  
Vol 27 ◽  
pp. 204020661988922 ◽  
Author(s):  
Francesco Di Palma ◽  
Gian Luca Daino ◽  
Venkata Krishnan Ramaswamy ◽  
Angela Corona ◽  
Aldo Frau ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Type I Interferon ◽  
Coiled Coil ◽  
Coarse Grained ◽  
Type I ◽  
Wild Type ◽  
Terminal Domain ◽  
Coiled Coil Region ◽  
Interferon Antagonism

Ebola virus high lethality relies on its ability to efficiently bypass the host innate antiviral response, which senses the viral dsRNA through the RIG-I receptor and induces type I interferon α/β production. In the bypassing action, the Ebola virus protein VP35 plays a pivotal role at multiple levels of the RIG-I cascade, masking the viral 5′-triphosphorylated dsRNA from RIG-I, and interacting with other cascade components. The VP35 type I interferon inhibition is exerted by the C-terminal domain, while the N-terminal domain, containing a coiled-coil region, is primarily required for oligomerization. However, mutations at key VP35 residues L90/93/107A (VP35-3m) in the coiled-coil region were reported to affect oligomerization and reduce type I interferon antagonism, indicating a possible but unclear role of homo-oligomerization on VP35 interaction with the RIG-I pathway components. In this work, we investigated the VP35 dimerization thermodynamics and its contribution to type I interferon antagonism by computational and biological methods. Focusing on the coiled-coil region, we combined coarse-grained and all-atom simulations on wild type VP35 and VP35-3m homo-dimerization. According to our results, wild type VP35 coiled-coil is able to self-assemble into dimers, while VP35-3m coiled-coil shows poor propensity to even dimerize. Free-energy calculations confirmed the key role of L90, L93 and L107 in stabilizing the coiled-coil homo-dimeric structure. In vitro type I interferon antagonism studies, using full-length wild type VP35 and VP35-3m, revealed that VP35 homo-dimerization is an essential preliminary step for dsRNA binding, which appears to be the main factor of the VP35 RIG-I cascade inhibition, while it is not essential to block the other steps.

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