Interaction between human tRNA synthetases involves repeated sequence elements.

Rho, Seung Bae; Lee, Kyoung Hoa; Kim, Jung Ho; Shiba, Kiyotaka; Jo, Yeong Joon; Kim, Sung Hoon

doi:10.1073/pnas.93.19.10128

Cited by 81 publications

(88 citation statements)

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“…A positive interaction was defined as an ability to support cell growth on leucinedepleted media or by a blue result by white color testing on X-galcontaining media (22). DNAs encoding human TB10 WT or T20S mutant (Thr 20 to Ser 20 mutation) for in vitro binding assay was ligated into pGEX4T-1 (Amersham Biosciences) using EcoRI and SalI to generate glutathione S-transferase (GST) fusion protein.…”

Section: Methodsmentioning

confidence: 99%

“…Ovarian Cancer CellsWe tested the contribution made by individual amino acids in the 17 LKKTET 22 motif to the biological activity of TB10 by Ala and Ser scanning mutagenesis. Each of the mutant sequences was confirmed by automatic sequencing, and transfected to SKOV-3 and PA-1 (data not shown) ovarian cancer cells.…”

Section: Effect Of Tb10 Point Mutations On Growth Inmentioning

confidence: 99%

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The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

Rho

Lee

Chun

et al. 2005

Journal of Biological Chemistry

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Thymosin ␤-10 (TB10) is an actin monomer-sequestering peptide that consists of 43 amino acid residues and that tends to form ␣-helical structures. Previously, we showed that the overexpression of TB10 dramatically increases the frequency of apoptosis in human ovarian cancer cells. To identify the critical residues responsible for TB10-mediated apoptosis, we used a series of computational methods. First, a three-dimensional structure of human TB10 was constructed using the homology modeling method with the calf thymosin ␤-9 NMR structure as a template. Although the sequences of both of these structures are almost identical, 200-ps molecular dynamics simulation results showed that their secondary structures differ. Analyses of molecular dynamics snapshot structures suggested that the TB10 structure is conformationally more complicated than the TB9 structure. The conserved 17 LKKTET 22 motif region of TB10 was tested by Ala and Ser scanning mutagenesis using computational and biochemical methods, and 12 mutants were transfected into cancer cell lines and tested for their effects on growth arrest. Of the 12 mutants examined, only the Thr 20 to Ser 20 mutation showed reduced growth arrest. These results strongly suggest that Thr 20 is specifically required for actin sequestration by TB10 in ovarian cancer cells. These results may provide useful information for the development of a new ovarian cancer therapy.The ␤-thymosins are small (molecular mass Ͻ 5 kDa) highly conserved acidic proteins. They were originally identified in calf thymus and thought to be involved in immunomodulatory functions (1). At least 16 ␤-thymosin isoforms have been identified from various vertebrates and invertebrates, and most, including thymosin ␤-4 (TB4), 3 are constitutively expressed in most tissues (2-5). A majority share a conserved hexapeptide motif, "LKKTET," which is critical for binding actin, and have important roles in maintaining cellular functions, such as cell morphology, proliferation, and locomotion (6). In most mammalian tissues, TB4 and TB10 are major actin monomer-sequestering proteins (7,8). Both form a 1:1 complex with G-actin and inhibit its polymerization to F-actin (9 -11).TB10 is more selectively modulated during embryological development than TB4 and is associated with several diseases (12-15). In addition, TB10 up-regulates the anti-apoptotic protein Bcl-2, which is associated with neoplastic transformation (16). Thymosin ␤-9 (TB9) overexpression was also found to modulate infected bovine macrophage apoptosis (17).In the present study, we employed a combined approach involving molecular modeling and site-directed mutagenesis to better understand the molecular regions of TB10 that control apoptosis. On the basis of its computer model-determined three-dimensional structure, we identified a region of TB10 that might be critical for apoptotic induction. This region was then tested in transfected ovarian cancer cell lines via mutagenesis (Ala and Ser scanning) of the conserved 17 LKKTET 22 motif with respect to actin bi...

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Section: Methodsmentioning

confidence: 99%

Section: Effect Of Tb10 Point Mutations On Growth Inmentioning

confidence: 99%

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

Rho

Lee

Chun

et al. 2005

Journal of Biological Chemistry

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“…Given the variety of functions that have been associated with aminoacyl-tRNA synthetases (46), sumoylation of these enzymes could help mediate the stress response in any number of ways. For example, both EPRS and MRS are components of the MSC, and the rep domain, which is the region of EPRS targeted for sumoylation, is required for the formation of this complex (67). The functional significance of the MSC is unclear, although it may play a role in the trafficking of tRNA from the nucleus to the ribosome (47).…”

Section: Sumo Deconjugation As a Means Tomentioning

confidence: 99%

Drosophila Ulp1, a Nuclear Pore-associated SUMO Protease, Prevents Accumulation of Cytoplasmic SUMO Conjugates

Smith

Bhaskar

Fernandez

et al. 2004

Journal of Biological Chemistry

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SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substratespecific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.The regulation of protein function by reversible post-translational modification is an effective means for regulating protein stability, activity, subcellular localization, and interaction specificity. One such modification involves the covalent attachment of the small ubiquitin-like modifier protein (SUMO) 1 (also called Smt3), to its target proteins via a series of enzymatic steps that closely resemble the reactions of the ubiquitylation pathway (1-3).Despite the sequence and structural homology between SUMO and ubiquitin, it is clear that sumoylation and ubiquitylation have distinct functional consequences. Ubiquitylation most frequently results in degradation of the substrate protein through targeting to the 26 S proteasome (4). The consequences of sumoylation are diverse and target-specific, with no evidence to suggest a role in promoting protein degradation. Conjugation of SUMO to RanGAP1 is required to target it to the nuclear pore complex (NPC), where it makes a stable interaction with the Ran-binding protein RanBP2 (5-7). Sumoylation of PML is essential for its recruitment to discrete intranuclear foci called PML oncogenic domains (PODs) (8 -11). For some sumoylation targets, including Smad4 and IB␣, this modification appears to increase protein stability by blocking ubiquitin-mediated degradation (12, 13). Finally, an increasing number of DNA binding transcription factors have been identified as substrates for sumoylation (14,15). Sumoylation of transcription factors can alter their activity in multiple ways. For example, in the ca...

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“…The underlying basis of association has been demonstrated for several of the polypeptides to be the hydrophobic nature of extensions unique to the eukaryotic enzymes (reviewed in Mirande, 1991;Yang, 1996). In addition, specific sequence motifs have been identified in synthetase polypeptides and p l 8 that play a role in proteinprotein interaction (Quevillon & Mirande, 1996;Rho et al, 1996). The overall shape and size of the particle are known from electron microscopy studies (Norcum, 1989).…”

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confidence: 99%

Structural analysis of the multienzyme aminoacyl‐tRNA synthetase complex: A three‐domain model based on reversible chemical crosslinking

Norcum

Warrington

1998

Protein Science

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A subset of eukaryotic aminoacyl-tRNA synthetases (a-RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two-dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K-RS:p38, D-RS:K-RS, R-RS dimer, K-RS dimer, K-RS:Q-RS, E/P-RS:K-RS, E/P-RS:I-RS, and Q-RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D-RS dimer, R-RS:p43, D-RS:Q-RS, D-RS:M-RS, K-RS:L-RS, I-RS:R-RS, D-RS:E/P-RS, I-RS:Q-RS, I-RS:L-RS. One trimeric association is seen: E/P-RS:I-RS:L-RS.The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl-tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U-shaped particle in which each ''arm'' is considered a domain and the third forms the "base" of the structure. The arms have been termed domain I (D-RS, M-RS, Q-RS) and domain I1 (K-RS, R-RS), with domain III (E/P-RS, I-RS, L-RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three-dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl-tRNA synthetase complex.Keywords: aminoacyl-tRNA synthetase complex; multienzyme complex; protein-protein interaction; protein structure; reversible chemical crosslinking Aminoacyl-tRNA synthetases are a family of enzymes characterized by their role in coupling each amino acid with the correct tRNA for subsequent use in protein biosynthesis. In addition to this fundamental role in translation, these enzymes are widely studied as models for the evolution of modular proteins and for insight into the mechanisms of RNA-protein recognition. These enzymes also have additional cellular functions, such as synthesis of signaling dinucleotides. There are several recent reviews of these topics (Mirande, 1991;Schulman, 1991;Cusack, 1995).One of the most intriguing and least understood properties of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multienzyme complex of a subset Reprint requests to: Mona T. Norcum, Biochemistry Department, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505; e-mail: mnorcum@fiona.umsmed.edu.Abbreviations: a-RS, aminoacyl-tRNA synthetase, a is the one-letter ambo acid abbreviation; cis-DDP, cis-dichlorodiammine platinum; EGS, erhylene glycobis(succinimydy1succinate); SMPT, 4-succinimydyl oxycarbonyl-2-pyridyldithiotoluene. of these proteins (reviewed in Kisselev & Wolfson, 1994;Yang, 1996). As isolated fr...

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Interaction between human tRNA synthetases involves repeated sequence elements.

Cited by 81 publications

References 50 publications

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

The Identification of Apoptosis-related Residues in Human Thymosin β-10 by Mutational Analysis and Computational Modeling

Drosophila Ulp1, a Nuclear Pore-associated SUMO Protease, Prevents Accumulation of Cytoplasmic SUMO Conjugates

Structural analysis of the multienzyme aminoacyl‐tRNA synthetase complex: A three‐domain model based on reversible chemical crosslinking

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