Ubiquitylation is a critical post-translational modification that controls a wide variety of proc... more Ubiquitylation is a critical post-translational modification that controls a wide variety of processes in eukaryotes. Ubiquitin chains of different topologies are specialized for different cellular functions and control the stability, activity, interaction properties, and localization of many different proteins. Recent work has highlighted a role for branched ubiquitin chains in the regulation of cell signaling and protein degradation pathways. Similar to their unbranched counterparts, branched ubiquitin chains are remarkably diverse in terms of their chemical linkages, structures, and the biological information they transmit. In this review, we discuss emerging themes related to the architecture, synthesis, and functions of branched ubiquitin chains. We also describe methodologies that have recently been developed to identify and decode the functions of these branched polymers.
The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G cyclins ... more The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with ...
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attri... more mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We ...
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attri... more mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We ...
How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss... more How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial ma...
the Grb2, p130cas, and Nck adaptor proteins. ofadhesion kinase-c-Src complex: involvement Fibrone... more the Grb2, p130cas, and Nck adaptor proteins. ofadhesion kinase-c-Src complex: involvement Fibronectin-stimulated signaling from a focal
Proceedings of the National Academy of Sciences, 2021
Significance Phosphohistidine (pHis) is a labile posttranslational modification with two isoforms... more Significance Phosphohistidine (pHis) is a labile posttranslational modification with two isoforms, 1-pHis and 3-pHis, involved in many cellular processes across the kingdoms of life. Due to its lability, it is difficult to study the pHis modification using standard biochemical tools and techniques. Previously, we developed monoclonal antibodies (mAbs) against the 1-pHis and 3-pHis modifications using stable phosphotriazolylalanine mimetics as immunogens. These antibodies are promising tools to uncover the role of pHis in eukaryotic cells. Here, we report the crystal structures of five of these mAbs bound to their cognate phosphotriazolylalanine peptides, thus providing insight into the structure–function relationships that guide pHis recognition and establishing a foundation for the structure-guided design of improved pHis antibodies.
The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), we... more The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylat...
We have compared the polypeptide products of the src gene of several strains of Rous sarcoma viru... more We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides result...
Ubiquitylation is a critical post-translational modification that controls a wide variety of proc... more Ubiquitylation is a critical post-translational modification that controls a wide variety of processes in eukaryotes. Ubiquitin chains of different topologies are specialized for different cellular functions and control the stability, activity, interaction properties, and localization of many different proteins. Recent work has highlighted a role for branched ubiquitin chains in the regulation of cell signaling and protein degradation pathways. Similar to their unbranched counterparts, branched ubiquitin chains are remarkably diverse in terms of their chemical linkages, structures, and the biological information they transmit. In this review, we discuss emerging themes related to the architecture, synthesis, and functions of branched ubiquitin chains. We also describe methodologies that have recently been developed to identify and decode the functions of these branched polymers.
The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G cyclins ... more The retinoblastoma gene product (Rb) can interact efficiently with two of three D-type G cyclins (D2 and D3) in vitro. Binding depended upon the minimal regions of Rb necessary for its growth-suppressive activity, as well as upon the D-type cyclin sequence motif shared with ...
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attri... more mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We ...
mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attri... more mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We ...
How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss... more How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial ma...
the Grb2, p130cas, and Nck adaptor proteins. ofadhesion kinase-c-Src complex: involvement Fibrone... more the Grb2, p130cas, and Nck adaptor proteins. ofadhesion kinase-c-Src complex: involvement Fibronectin-stimulated signaling from a focal
Proceedings of the National Academy of Sciences, 2021
Significance Phosphohistidine (pHis) is a labile posttranslational modification with two isoforms... more Significance Phosphohistidine (pHis) is a labile posttranslational modification with two isoforms, 1-pHis and 3-pHis, involved in many cellular processes across the kingdoms of life. Due to its lability, it is difficult to study the pHis modification using standard biochemical tools and techniques. Previously, we developed monoclonal antibodies (mAbs) against the 1-pHis and 3-pHis modifications using stable phosphotriazolylalanine mimetics as immunogens. These antibodies are promising tools to uncover the role of pHis in eukaryotic cells. Here, we report the crystal structures of five of these mAbs bound to their cognate phosphotriazolylalanine peptides, thus providing insight into the structure–function relationships that guide pHis recognition and establishing a foundation for the structure-guided design of improved pHis antibodies.
The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), we... more The NME (Non-metastatic) family members, also known as NDPKs (nucleoside diphosphate kinases), were originally identified and studied for their nucleoside diphosphate kinase activities. This family of kinases is extremely well conserved through evolution, being found in prokaryotes and eukaryotes, but also diverges enough to create a range of complexity, with homologous members having distinct functions in cells. In addition to nucleoside diphosphate kinase activity, some family members are reported to possess protein-histidine kinase activity, which, because of the lability of phosphohistidine, has been difficult to study due to the experimental challenges and lack of molecular tools. However, over the past few years, new methods to investigate this unstable modification and histidine kinase activity have been reported and scientific interest in this area is growing rapidly. This review presents a global overview of our current knowledge of the NME family and histidine phosphorylat...
We have compared the polypeptide products of the src gene of several strains of Rous sarcoma viru... more We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides result...
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Papers by Tony Hunter