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Marc Ladanyi - One of the best experts on this subject based on the ideXlab platform.
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expression of f actin capping Protein Subunit beta capzb is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Midori Todaishii, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Marc LadanyiAbstract:Background A previous proteomics study demonstrated the overexpression of F-actin capping Protein Subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.
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expression of f actin capping Protein Subunit beta capzb is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Midori Todaishii, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Marc LadanyiAbstract:A previous proteomics study demonstrated the overexpression of F-actin capping Protein Subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS. Cellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative Protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific Proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained Protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 Protein using INI1-overexpressing EpiS cells. All 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of Protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 Proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression. CAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoProtein CAPZB in the pathogenesis of EpiS remains to be clarified.
Treena Cranston - One of the best experts on this subject based on the ideXlab platform.
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activating mutations of the g Protein Subunit α11 interdomain interface cause autosomal dominant hypocalcemia type 2
The Journal of Clinical Endocrinology and Metabolism, 2020Co-Authors: Caroline M Gorvin, Treena Cranston, Victoria Stokes, Hannah Boon, Anna K Gluck, Shailini Bahl, Tessa Homfray, Theingi Aung, Brian Shine, Kate E LinesAbstract:CONTEXT Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and ADH2) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-Protein Subunit α 11 (Gα 11), respectively. More than 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function Gα 11 mutations are reported to date. METHODS We ascertained 2 additional ADH families and investigated them for CaSR and Gα 11 mutations. The effects of identified variants on CaSR signaling were evaluated by transiently transfecting wild-type (WT) and variant expression constructs into HEK293 cells stably expressing CaSR (HEK-CaSR), and measuring intracellular calcium (Ca2+i) and MAPK responses following stimulation with extracellular calcium (Ca2+e). RESULTS CaSR variants were not found, but 2 novel heterozygous germline Gα 11 variants, p.Gly66Ser and p.Arg149His, were identified. Homology modeling of these revealed that the Gly66 and Arg149 residues are located at the interface between the Gα 11 helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of 3 other reported ADH2 mutations. The Ca2+i and MAPK responses of cells expressing the variant Ser66 or His149 Gα 11 Proteins were similar to WT cells at low Ca2+e, but significantly increased in a dose-dependent manner following Ca2+e stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function Gα 11 mutations. Treatment of Ser66- and His149-Gα 11 expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized Ca2+i and MAPK responses. CONCLUSION Two novel ADH2-causing mutations that highlight the Gα 11 interdomain interface as a hotspot for gain-of-function Gα 11 mutations have been identified.
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identification of a g Protein Subunit α11 gain of function mutation val340met in a family with autosomal dominant hypocalcemia type 2 adh2
Journal of Bone and Mineral Research, 2016Co-Authors: Sian E Piret, Caroline M Gorvin, Alistair T Pagnamenta, Sarah A Howles, Treena Cranston, Nigel Rust, Andrew M Nesbit, Ben Glaser, Jenny C Taylor, Andreas E BuchsAbstract:Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain-of-function mutations of the G-Protein coupled calcium-sensing receptor (CaSR), and ADH2 caused by germline gain-of-function mutations of G-Protein Subunit α-11 (Gα11 ). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole-exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three-dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C-terminal α5 helix, which may affect G-Protein coupled receptor binding and G-Protein activation. In vitro functional expression of wild-type (Val340) and mutant (Met340) Gα11 Proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration-response curve with a significantly (p < 0.0001) reduced mean half-maximal concentration (EC50 ) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild-type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain-of-function mutation. A novel His403Gln variant in transforming growth factor, beta-induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain-of-function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C-terminal region for G-Protein function and CaSR signal transduction. © 2016 American Society for Bone and Mineral Research.
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Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia
The New England journal of medicine, 2013Co-Authors: M. Andrew Nesbit, Sarah A Howles, Treena Cranston, Nigel Rust, Fadil Hannan, Valerie Babinsky, Rosie A. Head, Maurine R. Hobbs, Hunter Heath, Rajesh V ThakkerAbstract:Background Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding Protein (G-Protein)–coupled receptor that signals through the G-Protein Subunit α11 (Gα11). Type 3 is associated with adaptor-related Protein complex 2, sigma 1 Subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. Methods We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalc...
Kenta Mukaihara - One of the best experts on this subject based on the ideXlab platform.
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expression of f actin capping Protein Subunit beta capzb is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Midori Todaishii, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Marc LadanyiAbstract:Background A previous proteomics study demonstrated the overexpression of F-actin capping Protein Subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.
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expression of f actin capping Protein Subunit beta capzb is associated with cell growth and motility in epithelioid sarcoma
BMC Cancer, 2016Co-Authors: Kenta Mukaihara, Yoshiyuki Suehara, Shinji Kohsaka, Daisuke Kubota, Midori Todaishii, Keisuke Akaike, Tsutomu Fujimura, Eisuke Kobayashi, Takashi Yao, Marc LadanyiAbstract:A previous proteomics study demonstrated the overexpression of F-actin capping Protein Subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS. Cellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative Protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific Proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained Protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 Protein using INI1-overexpressing EpiS cells. All 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of Protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 Proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression. CAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoProtein CAPZB in the pathogenesis of EpiS remains to be clarified.
Michael E Harris - One of the best experts on this subject based on the ideXlab platform.
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the contribution of the c5 Protein Subunit of escherichia coli ribonuclease p to specificity for precursor trna is modulated by proximal 5 leader sequences
RNA, 2017Co-Authors: Courtney N Niland, David R Anderson, Eckhard Jankowsky, Michael E HarrisAbstract:Recognition of RNA by RNA processing enzymes and RNA binding Proteins often involves cooperation between multiple Subunits. However, the interdependent contributions of RNA and Protein Subunits to molecular recognition by ribonucleoProteins are relatively unexplored. RNase P is an endonuclease that removes 5' leaders from precursor tRNAs and functions in bacteria as a dimer formed by a catalytic RNA Subunit (P RNA) and a Protein Subunit (C5 in E. coli). The P RNA Subunit contacts the tRNA body and proximal 5' leader sequences [N(-1) and N(-2)] while C5 binds distal 5' leader sequences [N(-3) to N(-6)]. To determine whether the contacts formed by P RNA and C5 contribute independently to specificity or exhibit cooperativity or anti-cooperativity, we compared the relative kcat/Km values for all possible combinations of the six proximal 5' leader nucleotides (n = 4096) for processing by the E. coli P RNA Subunit alone and by the RNase P holoenzyme. We observed that while the P RNA Subunit shows specificity for 5' leader nucleotides N(-2) and N(-1), the presence of the C5 Protein reduces the contribution of P RNA to specificity, but changes specificity at N(-2) and N(-3). The results reveal that the contribution of C5 Protein to RNase P processing is controlled by the identity of N(-2) in the pre-tRNA 5' leader. The data also clearly show that pairing of the 5' leader with the 3' ACCA of tRNA acts as an anti-determinant for RNase P cleavage. Comparative analysis of genomically encoded E. coli tRNAs reveals that both anti-determinants are subject to negative selection in vivo.
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analysis of the rna binding specificity landscape of c5 Protein reveals structure and sequence preferences that direct rnase p specificity
Chemistry & Biology, 2016Co-Authors: Hsuan Chun Lin, Courtney N Niland, Eckhard Jankowsky, Jing Zhao, Brandon Tran, Michael E HarrisAbstract:RNA binding Proteins (RBPs) are typically involved in non-equilibrium cellular processes, and specificity can arise from differences in ground state, transition state, or product states of the binding reactions for alternative RNAs. Here, we use high-throughput methods to measure and analyze the RNA association kinetics and equilibrium binding affinity for all possible sequence combinations in the precursor tRNA binding site of C5, the essential Protein Subunit of Escherichia coli RNase P. The results show that the RNA sequence specificity of C5 arises due to favorable RNA-Protein interactions that stabilize the transition state for association and bound enzyme-substrate complex. Specificity is further impacted by unfavorable RNA structure involving the C5 binding site in the ground state. The results illustrate a comprehensive quantitative approach for analysis of RNA binding specificity, and show how both RNA structure and sequence preferences of an essential Protein Subunit direct the specificity of a ribonucleoProtein enzyme.
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evidence that substrate specific effects of c5 Protein lead to uniformity in binding and catalysis by rnase p
The EMBO Journal, 2006Co-Authors: Lei Sun, Frank E Campbell, Nathan H Zahler, Michael E HarrisAbstract:The ribonucleoProtein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the Protein Subunit to 5′ leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5′ leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (>900-fold) contributions of the Protein Subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P Protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.
Venkat Gopalan - One of the best experts on this subject based on the ideXlab platform.
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the rice rnase p Protein Subunit rpp30 confers broad spectrum resistance to fungal and bacterial pathogens
Plant Biotechnology Journal, 2021Co-Authors: Yehui Xiong, Venkat Gopalan, Lien B Lai, Kai Zhang, Houxiang Kang, Liangying Dai, Guoliang Wang, Wende LiuAbstract:RNase P functions either as a catalytic ribonucleoProtein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP Subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This Protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars.
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A functional RNase P Protein Subunit of bacterial origin in some eukaryotes
Molecular Genetics and Genomics, 2011Co-Authors: Pilar Bernal-bayard, Venkat Gopalan, Gireesha Mohannath, Agustin VioqueAbstract:RNase P catalyzes 5′-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a Protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten Proteins, all unrelated to the bacterial Protein. Unexpectedly, a nuclear-encoded bacterial RNase P Protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri . We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter’s presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5′-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri , the smallest known free-living eukaryote.
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structure of mth11 mth rpp29 an essential Protein Subunit of archaeal and eukaryotic rnase p
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: William P Boomershine, Venkat Gopalan, Craig A Mcelroy, Hsinyue Tsai, Ross C Wilson, Mark P FosterAbstract:We have determined the solution structure of Mth11 (Mth Rpp29), an essential Subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitous ribonucleoProtein enzyme primarily responsible for cleaving the 5′ leader sequence during maturation of tRNAs in all three domains of life. In eubacteria, this enzyme is made up of two Subunits: a large RNA (≈120 kDa) responsible for mediating catalysis, and a small Protein cofactor (≈15 kDa) that modulates substrate recognition and is required for efficient in vivo catalysis. In contrast, multiple Proteins are associated with eukaryotic and archaeal RNase P, and these Proteins exhibit no recognizable homology to the conserved bacterial Protein Subunit. In reconstitution experiments with recombinantly expressed and purified Protein Subunits, we found that Mth Rpp29, a homolog of the Rpp29 Protein Subunit from eukaryotic RNase P, is an essential Protein component of the archaeal holoenzyme. Consistent with its role in mediating Protein–RNA interactions, we report that Mth Rpp29 is a member of the oligonucleotide/oligosaccharide binding fold family. In addition to a structured β-barrel core, it possesses unstructured N- and C-terminal extensions bearing several highly conserved amino acid residues. To identify possible RNA contacts in the Protein–RNA complex, we examined the interaction of the 11-kDa Protein with the full 100-kDa Mth RNA Subunit by using NMR chemical shift perturbation. Our findings represent a critical step toward a structural model of the RNase P holoenzyme from archaebacteria and higher organisms.
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elucidation of structure function relationships in the Protein Subunit of bacterial rnase p using a genetic complementation approach
Nucleic Acids Research, 2002Co-Authors: Milan Jovanovic, Ruth Sanchez, Sidney Altman, Venkat GopalanAbstract:RNase P is a ribonucleoProtein involved in tRNA biosynthesis in all living organisms. Bacterial RNase P is comprised of a catalytic RNA Subunit and a lone Protein cofactor which plays a supporting, albeit essential, role in the tRNA processing reaction in vivo. In this study, we have searched various databases to identify homologs of the Protein Subunit of RNase P from diverse bacteria and used an alignment of their primary sequences to determine the most highly conserved residues, and thereby extend earlier predictions of which residues might play an important role in RNA recognition. By employing a genetic complementation assay, we have also gained insights into structure- function relationships in the Protein Subunit of bacterial RNase P.
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mapping rna Protein interactions in ribonuclease p from escherichia coli using disulfide linked edta fe
Journal of Molecular Biology, 2000Co-Authors: Roopa Biswas, Sidney Altman, David W Ledman, Robert O Fox, Venkat GopalanAbstract:The Protein Subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe). This reaction converts C5 Protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA Subunit of E. coli RNase P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 Protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 Protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-Protein interface in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and Protein Subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P.
