Leucine zipper domain of 52 kDa SS-A/Ro promotes protein dimer formation and inhibits in vitro transcription activity

Wang, Dunrui; Buyon, Jill P.; Yang, Zhenguo; Donato, Francis Di; Miranda-Carús, María-Eugenia; Chan, Edward K. L.

doi:10.1016/s0304-4165(01)00212-4

Cited by 24 publications

(18 citation statements)

References 41 publications

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“…This conclusion was again based upon the electrophoretic mobility of chemically cross-linked TRIM21 proteins, however, which may be subject to the same uncertainties as cross-linked TRIM5␣ proteins. Moreover, another group has reported that the TRIM21/SS-A/Ro52 protein may instead be dimeric, based on its gel filtration mobility (59), and two groups have reported that the isolated coiled-coil region of TRIM21 forms dimers rather than trimers (27,40). We therefore speculate that dimerization may be a common property of many members of the tripartite motif protein family.…”

Section: Discussionmentioning

confidence: 70%

Biochemical Characterization of a Recombinant TRIM5α Protein That Restricts Human Immunodeficiency Virus Type 1 Replication

Langelier

Sandrin

Eckert

et al. 2008

J Virol

113

161

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The rhesus monkey intrinsic immunity factor TRIM5␣ rh recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5␣ proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5␣ rh protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5␣ proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5␣ proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5␣ proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.Susceptibility to retroviral infections influences species survival and has driven the evolution of cellular restriction factors that inhibit retroviral replication. One important antiretroviral intrinsic immune response is mediated by TRIM5␣, which can block early postentry steps in the replication of certain retroviruses in specific primate lineages (3, 37, 55, 58). Under normal restrictive conditions, TRIM5␣ proteins block accumulation of retroviral reverse transcripts (55) and accelerate the rate at which viral capsids dissociate from high-molecularweight complexes into lower-molecular-weight subunits (42,56). The allelic specificity of TRIM5␣ restriction is illustrated by the fact that rhesus macaque TRIM5␣ potently inhibits human immunodeficiency virus type 1 (HIV-1) replication, whereas human TRIM5␣ instead exhibits restriction activity against N-tropic murine leukemia virus but not 29,43,55,67). These differences can be attributed to the differential abilities of TRIM5␣ proteins to interact with retroviral capsids after viral entry (34,42,56).Like other tripartite (TRIM) family members, TRIM5␣ contains RING, B-box, coiled-coil, and B30.2/SPRY domains, and each of t...

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Section: Discussionmentioning

confidence: 70%

Biochemical Characterization of a Recombinant TRIM5α Protein That Restricts Human Immunodeficiency Virus Type 1 Replication

Langelier

Sandrin

Eckert

et al. 2008

J Virol

113

161

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“…The 52-␣ form is ubiquitously expressed, whereas 52-␤, lacking the central leucine zipper domain, has been detected at higher levels than 52-␣ in fetal heart aged 14 -16 wk of gestation (29). The 52-␤ protein was shown to be present as a monomer, and to have promoted transcription activity in vitro (30). Therefore, the 52-␤ protein is considered to play a role in specific gene activation related to cardiac development, although the target gene(s) is not identified.…”

Section: Discussionmentioning

confidence: 97%

SS-A/Ro52, an Autoantigen Involved in CD28-Mediated IL-2 Production

Ishii

Ohnuma

Murakami

et al. 2003

The Journal of Immunology

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An autoantibody against SS-A/Ro52 (Ro52) is most frequently found in the sera of patients with Sjögren’s syndrome, systemic lupus erythematosus, and congenital heart block from anti-Ro52 Ab-positive mother. However, the physiological function of the autoantigen SS-A/Ro52 has not yet been elucidated. In this study, we describe the role of Ro52 protein in T cell activation. Overexpression of SS-A/Ro52 in Jurkat T cell resulted in enhanced IL-2 production following CD28 stimulation. Furthermore, transfection of anti-Ro52-specific small RNA duplexes partially blocked the expression of native and overexpressed Ro52 in Jurkat T cell, resulting in decreased IL-2 production via CD28 pathway in these cells. Finally, intracellular localization of Ro52 dramatically changed following CD28 stimulation. Our data reveal a novel function of Ro52 in CD28-mediated pathway, which eventually contributes to cytokine production and expression of the T cell biological programs.

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“…At the C terminus of the Ro52 RBCC region, a well conserved B30.2 domain is not yet associated with any defined function (13,14). It has been suggested that Ro52 is involved in transcriptional regulation (15,16), and mono-ubiquitination of Ro52 (17), as well as interaction with a deubiquitinating enzyme, has been shown (18). Still, the function of Ro52 remains to be defined and might shed light on its targeting in autoimmune disease.…”

mentioning

confidence: 99%

Structural Organization and Zn2+-dependent Subdomain Interactions Involving Autoantigenic Epitopes in the Ring-B-box-Coiled-coil (RBCC) Region of Ro52

Hennig

Ottosson

Andrésen

et al. 2005

Journal of Biological Chemistry

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Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn 2؉ -binding Ring-Bbox region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn 2؉ -binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn 2؉ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities.Ro52 is one of the main autoantigens in Sjögren syndrome, but autoantibodies to this intracellular protein occur also in systemic lupus erythematosus and rheumatoid arthritis (1). The immunodominant epitopes in Ro52 are predominantly localized in the structurally stable regions (2). The Ro52 coiled-coil contains a putative leucine-zipper between residues 211 and 232. Patient-derived monoclonal antibodies that bind to a peptide representing residues 200 -239 (p200) of the Ro52 protein cause accumulating intracellular calcium levels in neonatal cardiomyocytes and relate to the development of congenital heart block (3-5). Another antigenic epitope has been mapped to the N-terminal Zn 2ϩ -binding region of Ro52 (6) and was also suggested to relate to development of congenital heart block (7). However, the detailed location of the epitope in this 15-kDa region is yet unknown. The epitope is presumably conformation-dependent, because antigenicity is only observed in the reduced state (6), and because cysteines are conserved in the suggested Zn 2ϩ -binding sites. The reason why Ro52 is targeted in autoimmune disease is not known to date. Ro52 belongs to the rapidly growing RING/B-box/coiled-coil (RBCC) 2 family, also denoted as TRIM (trip...

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Leucine zipper domain of 52 kDa SS-A/Ro promotes protein dimer formation and inhibits in vitro transcription activity

Cited by 24 publications

References 41 publications

Biochemical Characterization of a Recombinant TRIM5α Protein That Restricts Human Immunodeficiency Virus Type 1 Replication

Biochemical Characterization of a Recombinant TRIM5α Protein That Restricts Human Immunodeficiency Virus Type 1 Replication

SS-A/Ro52, an Autoantigen Involved in CD28-Mediated IL-2 Production

Structural Organization and Zn2+-dependent Subdomain Interactions Involving Autoantigenic Epitopes in the Ring-B-box-Coiled-coil (RBCC) Region of Ro52

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