The Experts below are selected from a list of 4680 Experts worldwide ranked by ideXlab platform
Eugene G. Mueller - One of the best experts on this subject based on the ideXlab platform.
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the pseudouridine synthases revisiting a mechanism that seemed settled
Journal of the American Chemical Society, 2004Co-Authors: Christopher J. Spedaliere, Joy M Ginter, Murray V Johnston, Eugene G. MuellerAbstract:RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential Aspartic Acid Residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the Aspartic Acid Residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The Aspartic Acid Residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.
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Critical Aspartic Acid Residues in Pseudouridine Synthases
Journal of Biological Chemistry, 1999Co-Authors: Vidhyashankar Ramamurthy, Steven L. Swann, Jennifer L. Paulson, Christopher J. Spedaliere, Eugene G. MuellerAbstract:Abstract The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411–2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996)Nucleic Acids Res. 24, 3756–3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998)Biochemistry 37, 344–351), the Santi group discovered that a single Aspartic Acid Residue is the only amino Acid present in all of the aligned sequences; they then demonstrated that this Aspartic Acid Residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the Aspartic Acid Residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned Aspartic Acid Residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned Aspartic Acid Residue might be a prerequisite for pseudouridine synthase function.
Isabel W. C. E. Arends - One of the best experts on this subject based on the ideXlab platform.
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Identification of catalytically important Residues of the carotenoid 1,2-hydratases from Rubrivivax gelatinosus and Thiocapsa roseopersicina
Applied microbiology and biotechnology, 2015Co-Authors: Aida Hiseni, Linda G. Otten, Isabel W. C. E. ArendsAbstract:Carotenoid 1,2-hydratases (CrtC) catalyze the selective addition of water to an isolated carbon–carbon double bond. Although their involvement in the carotenoid biosynthetic pathway is well understood, little is known about the mechanism by which these hydratases transform carotenoids such as lycopene into the corresponding hydroxyl compounds. Key Residues were identified at positions His239, Trp241, Tyr266, and Asp268 in CrtC from Rubrivivax gelatinosus (and corresponding positions in Thiocapsa roseopersicina). Alanine mutants at these positions were found to be completely inactive, suggesting their direct involvement in the catalytic reaction. Our resulting mechanistic hypothesis is in analogy with the recently studied class of terpenoid cyclase enzymes containing a highly Acidic Aspartic Residue in their active site. We propose that a similar Aspartic Acid Residue, which is conserved through all putative CrtCs, is involved in initial protonation of the double bond in lycopene.
Anne Mariecardine - One of the best experts on this subject based on the ideXlab platform.
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efficient therapeutic delivery by a novel cell penetrating peptide derived from acinus
Cancers, 2020Co-Authors: Justine Habault, Claire Fraser, Ewa Pasquereaukotula, Maelys Bornbony, Anne MariecardineAbstract:In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an Aspartic Acid Residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (Residues 377-399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sezary patients' circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sezary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.
Ralf G. Dietzgen - One of the best experts on this subject based on the ideXlab platform.
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Cytorhabdovirus P3 genes encode 30K-like cell-to-cell movement proteins.
Virology, 2016Co-Authors: Krin S. Mann, Nicolás Bejerman, Karyn N. Johnson, Ralf G. DietzgenAbstract:Plant viruses encode movement proteins (MP) to facilitate cell-to-cell transport through plasmodesmata. In this study, using trans-complementation of a movement-defective turnip vein-clearing tobamovirus (TVCV) replicon, we show for the first time for cytorhabdoviruses (lettuce necrotic yellows virus (LNYV) and alfalfa dwarf virus (ADV)) that their P3 proteins function as MP similar to the TVCV P30 protein. All three MP localized to plasmodesmata when ectopically expressed. In addition, we show that these MP belong to the 30K superfamily since movement was inhibited by mutation of an Aspartic Acid Residue in the critical 30K-specific LxD/N50-70G motif. We also report that Nicotiana benthamiana microtubule-associated VOZ1-like transcriptional activator interacts with LNYV P3 and TVCV P30 but not with ADV P3 or any of the MP point mutants. This host protein, which is known to interact with P3 of sonchus yellow net nucleorhabdovirus, may be involved in aiding the cell-to-cell movement of LNYV and TVCV.
John Frenz - One of the best experts on this subject based on the ideXlab platform.
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isomerization of an Aspartic Acid Residue in the complementarity determining regions of a recombinant antibody to human ige identification and effect on binding affinity
Biochemistry, 1996Co-Authors: Jerry Cacia, Rodney G Keck, Leonard G Presta, John FrenzAbstract:This report describes the effect on antigen binding of an isomerized aspartate Residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the Residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered Residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive Residue may ...
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isomerization of an Aspartic Acid Residue in the complementarity determining regions of a recombinant antibody to human ige identification and effect on binding affinity
Biochemistry, 1996Co-Authors: Jerry Cacia, Rodney G Keck, Leonard G Presta, John FrenzAbstract:This report describes the effect on antigen binding of an isomerized aspartate Residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the Residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered Residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive Residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies.
