{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:50153"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:50153","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Mechanistic insights into the PARP10-mediated ADP-ribosylation reaction and analysis of PARP10's subcellular localization","abstract":"ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono-ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. Accordingly, it is suggested to subdivide the PARP family into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases like PARP10, and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells. The transport across the nuclear envelope through nuclear pore complexes is achieved by the interaction of cargo proteins with karyopherins that mediate transport processes into and out of the nucleus. These interactions are in general dependent on the presence of a nuclear localization signal (NLS) or a nuclear export sequence (NES) in the cargo protein. It was shown previously that PARP10 possesses a functional NES and accumulates in the nucleus if nuclear export is inhibited. Since PARP10’s molecular weight exceeds the diffusion limit of the nuclear pore complexes significantly, it has to be imported into the nucleus in an active fashion. In order to locate a potential NLS in PARP10 subcellular localization studies and fluorescence bleaching techniques were performed to visualize nucleocytoplasmic shuttling. While it was possible to confirm the presence of a single classical NES in PARP10, no classical NLS could be detected. In fact the data suggest that three regions within PARP10 have the ability to enter the nucleus and thus potentially contribute to the nuclear import of PARP10. Surprisingly, the analysis of the subcellular PARP10 localization revealed the enrichment in remarkable cytoplasmic foci. Co-localization studies showed no conclusive overlap between PARP10 foci and previously described cytoplasmic substructures. Using time-lapse microscopy it was possible to show that PARP10 foci are highly dynamic, tend to fuse, can disintegrate and form de novo. Importantly, the deletion of the N-terminal RNA recognition motif in PARP10 dramatically impaired the formation of these foci indicating a potential role for RNA in their development. Although PARP10 foci do not overlap with RNA-processing particles, i.e. P bodies or stress granules, certain components present in P bodies are found in these foci. This co-localization is greatly enhanced in the presence of stress that blocks translation and triggers the formation of stress granules. Thus PARP10 enriches in novel cytoplasmic foci that might play a role in RNA processing suggesting a role for PARP10 in the regulation of cellular stress responses.","abstract_html":"ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono-ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. Accordingly, it is suggested to subdivide the PARP family into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases like PARP10, and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells. The transport across the nuclear envelope through nuclear pore complexes is achieved by the interaction of cargo proteins with karyopherins that mediate transport processes into and out of the nucleus. These interactions are in general dependent on the presence of a nuclear localization signal (NLS) or a nuclear export sequence (NES) in the cargo protein. It was shown previously that PARP10 possesses a functional NES and accumulates in the nucleus if nuclear export is inhibited. Since PARP10’s molecular weight exceeds the diffusion limit of the nuclear pore complexes significantly, it has to be imported into the nucleus in an active fashion. In order to locate a potential NLS in PARP10 subcellular localization studies and fluorescence bleaching techniques were performed to visualize nucleocytoplasmic shuttling. While it was possible to confirm the presence of a single classical NES in PARP10, no classical NLS could be detected. In fact the data suggest that three regions within PARP10 have the ability to enter the nucleus and thus potentially contribute to the nuclear import of PARP10. Surprisingly, the analysis of the subcellular PARP10 localization revealed the enrichment in remarkable cytoplasmic foci. Co-localization studies showed no conclusive overlap between PARP10 foci and previously described cytoplasmic substructures. Using time-lapse microscopy it was possible to show that PARP10 foci are highly dynamic, tend to fuse, can disintegrate and form de novo. Importantly, the deletion of the N-terminal RNA recognition motif in PARP10 dramatically impaired the formation of these foci indicating a potential role for RNA in their development. Although PARP10 foci do not overlap with RNA-processing particles, i.e. P bodies or stress granules, certain components present in P bodies are found in these foci. This co-localization is greatly enhanced in the presence of stress that blocks translation and triggers the formation of stress granules. Thus PARP10 enriches in novel cytoplasmic foci that might play a role in RNA processing suggesting a role for PARP10 in the regulation of cellular stress responses.","abstract_has_math":false,"creators":["Schuchlautz, Henning"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Lüscher, Bernhard"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2008,"date_issued":"2008","date_published":"2008","updated_at":"2026-07-30T19:40:16Z","subjects":["info:eu-repo/classification/ddc/570","NAD-ADP-Ribosyltransferase","Enzymkatalyse","ADP-Ribosylierung","Lokalisation","Enzym","RNS","Biowissenschaften, Biologie","PARP","mART","ADP-Ribose","Kern-Zytoplasma-Transport","RNA-Prozessierung","nucleocytoplasmic shuttling","RNA processing"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112708%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112708%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112708%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/50153","outbound_label":"Repository record","outbound_source":"dc:identifier"},"source_record":{"url":"https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50153","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Lüscher, Bernhard"]},{"key":"dc:creator","label":"Author","values":["Schuchlautz, Henning"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2008"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-24477"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","NAD-ADP-Ribosyltransferase","Enzymkatalyse","ADP-Ribosylierung","Lokalisation","Enzym","RNS","Biowissenschaften, Biologie","PARP","mART","ADP-Ribose","Kern-Zytoplasma-Transport","RNA-Prozessierung","nucleocytoplasmic shuttling","RNA processing"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/50153","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112708%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono-ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. Accordingly, it is suggested to subdivide the PARP family into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases like PARP10, and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells. The transport across the nuclear envelope through nuclear pore complexes is achieved by the interaction of cargo proteins with karyopherins that mediate transport processes into and out of the nucleus. These interactions are in general dependent on the presence of a nuclear localization signal (NLS) or a nuclear export sequence (NES) in the cargo protein. It was shown previously that PARP10 possesses a functional NES and accumulates in the nucleus if nuclear export is inhibited. Since PARP10’s molecular weight exceeds the diffusion limit of the nuclear pore complexes significantly, it has to be imported into the nucleus in an active fashion. In order to locate a potential NLS in PARP10 subcellular localization studies and fluorescence bleaching techniques were performed to visualize nucleocytoplasmic shuttling. While it was possible to confirm the presence of a single classical NES in PARP10, no classical NLS could be detected. In fact the data suggest that three regions within PARP10 have the ability to enter the nucleus and thus potentially contribute to the nuclear import of PARP10. Surprisingly, the analysis of the subcellular PARP10 localization revealed the enrichment in remarkable cytoplasmic foci. Co-localization studies showed no conclusive overlap between PARP10 foci and previously described cytoplasmic substructures. Using time-lapse microscopy it was possible to show that PARP10 foci are highly dynamic, tend to fuse, can disintegrate and form de novo. Importantly, the deletion of the N-terminal RNA recognition motif in PARP10 dramatically impaired the formation of these foci indicating a potential role for RNA in their development. Although PARP10 foci do not overlap with RNA-processing particles, i.e. P bodies or stress granules, certain components present in P bodies are found in these foci. This co-localization is greatly enhanced in the presence of stress that blocks translation and triggers the formation of stress granules. Thus PARP10 enriches in novel cytoplasmic foci that might play a role in RNA processing suggesting a role for PARP10 in the regulation of cellular stress responses."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University VI, 158 S. : Ill., graph. Darst. (2008). = Aachen, Techn. Hochsch., Diss., 2008"]},{"key":"dc:title","label":"Title","values":["Mechanistic insights into the PARP10-mediated ADP-ribosylation reaction and analysis of PARP10's subcellular localization"]}]}],"canonical_facts":{"dc:contributor":["Lüscher, Bernhard"],"dc:coverage":["DE"],"dc:creator":["Schuchlautz, Henning"],"dc:date":["2008"],"dc:description":["ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono-ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. Accordingly, it is suggested to subdivide the PARP family into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases like PARP10, and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells. The transport across the nuclear envelope through nuclear pore complexes is achieved by the interaction of cargo proteins with karyopherins that mediate transport processes into and out of the nucleus. These interactions are in general dependent on the presence of a nuclear localization signal (NLS) or a nuclear export sequence (NES) in the cargo protein. It was shown previously that PARP10 possesses a functional NES and accumulates in the nucleus if nuclear export is inhibited. Since PARP10’s molecular weight exceeds the diffusion limit of the nuclear pore complexes significantly, it has to be imported into the nucleus in an active fashion. In order to locate a potential NLS in PARP10 subcellular localization studies and fluorescence bleaching techniques were performed to visualize nucleocytoplasmic shuttling. While it was possible to confirm the presence of a single classical NES in PARP10, no classical NLS could be detected. In fact the data suggest that three regions within PARP10 have the ability to enter the nucleus and thus potentially contribute to the nuclear import of PARP10. Surprisingly, the analysis of the subcellular PARP10 localization revealed the enrichment in remarkable cytoplasmic foci. Co-localization studies showed no conclusive overlap between PARP10 foci and previously described cytoplasmic substructures. Using time-lapse microscopy it was possible to show that PARP10 foci are highly dynamic, tend to fuse, can disintegrate and form de novo. Importantly, the deletion of the N-terminal RNA recognition motif in PARP10 dramatically impaired the formation of these foci indicating a potential role for RNA in their development. Although PARP10 foci do not overlap with RNA-processing particles, i.e. P bodies or stress granules, certain components present in P bodies are found in these foci. This co-localization is greatly enhanced in the presence of stress that blocks translation and triggers the formation of stress granules. Thus PARP10 enriches in novel cytoplasmic foci that might play a role in RNA processing suggesting a role for PARP10 in the regulation of cellular stress responses."],"dc:identifier":["https://publications.rwth-aachen.de/record/50153","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112708%22"],"dc:language":["eng"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-24477"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University VI, 158 S. : Ill., graph. Darst. (2008). = Aachen, Techn. 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