Joseph Hinchey

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress. Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress. nuclear pore complex glutathione S-transferase green fluorescent protein phosphate-buffered saline polyacrylamide gel electrophoresis nuclear domain 10 SUMO-1 is a highly conserved, smallubiquitin-related modifier that has been shown to be covalently conjugated to a variety of cellular proteins (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Like ubiquitin, SUMO-1 is believed to form an isopeptide bond between the carboxyl terminus of SUMO-1 and a lysine side chain(s) of the target protein (2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar). The conjugation of SUMO-1 to cellular proteins has been implicated in multiple vital cellular processes, including nuclear transport, cell cycle control, oncogenesis, inflammation, and the response to virus infection (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 7.Saitoh H. Pu R.T. Dasso M. Trends Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 8.Hodges M. Tissot C. Freemont Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). has been that SUMO-1 conjugation to conjugation R.T. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google that SUMO-1 conjugation the target cellular R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google Scholar, M.J. EMBO J. 1998; PubMed Scopus Google known SUMO-1 in vertebrate the Ran GTPase-activating protein is the and is a highly protein that a nuclear protein for H. I. H. Sci. PubMed Scopus Google Scholar, F. Gerace L. Trends Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). has been demonstrated in and in that a lysine at in the terminus of is by SUMO-1 R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google Scholar). large of to be the nuclear a of in the nuclear pore complex (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, H. Pu R. M. Dasso M. Sci. 1997; PubMed Scopus Google Scholar). is in the that the conjugation of a ubiquitin-like at the domain target to the R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google cDNAs for proteins related to SUMO-1 have been human and and to as SUMO-2/SMT3A and (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, F. C. 1997; PubMed Scopus Google Scholar, H. Biochem. Biol. 1998; Google Scholar). in a wide of and cell that a role in cellular processes. of the of and -3 has revealed that SUMO-1 SUMO-2 and SUMO-2 and -3 is to SUMO-2 and -3 a SUMO-1. has been that SUMO-2 and -3 be to proteins in a to that of SUMO-1 when SUMO-2/3 was in cells T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This that SUMO-2/3 in a to that of SUMO-1. is the of SUMO-2/3 and that support cellular distinctions between two a of the role of SUMO-2/3 and the regulation of SUMO-2/3 SUMO-1 conjugation in vertebrate we have to SUMO-2/3 at the protein in We describe herein the of an antibody to SUMO-2 and this antibody and a we found that was by but by a of SUMO-2/3 than SUMO-1 in We demonstrated that the SUMO-2/3 conjugation be by protein-damaging This is the for the of a ubiquitin-like protein modification in cellular these we that there is regulation of SUMO-2/3 modification as SUMO-1 modification and that the SUMO-2/3 constitute an of the cellular response to environmental the of the known smallubiquitin-related be two in including human SUMO-1 and and the human and in for proteins to SUMO-2/3 revealed that SUMO-2/3 is highly not in vertebrate but in a role for the SUMO-2/3 conjugation a we demonstrated that and proteins in vertebrate cells. between SUMO-1 and was the of a large pool of non-conjugated SUMO-2/3 in cells. This of SUMO-2/3 to be for conjugation conjugation be induced by cellular such as acute and addition, known to the of proteins in the cells and to J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Cell. Biol. PubMed Scopus Google Scholar). Recently, we found that by the has been to protein Trends Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google the conjugation of SUMO-2/3 to a high molecular mass protein not We in the of proteins by SUMO-2/3 in a and the of such SUMO-2/3-modified proteins in has been that the to a wide variety of protein-damaging and environmental 15: PubMed Scopus Google Scholar). Intriguingly, M. J. Cell Biol. PubMed Scopus Google Scholar, EMBO J. PubMed Scopus Google and F. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google have been known to the quantitative of to the conjugated of the SUMO-2/3 conjugation in this that conjugation to cellular proteins of the by the of response in the SUMO-2/3 a by proteins identified and SUMO-2/3 conjugation the target protein by conjugation and the suggest that an for the be conjugated SUMO-1 R.T. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). conjugated SUMO-1 but subjected to of and is that these SUMO-2/3 conjugation the and the of such protein by by SUMO-1 has not been to protein results suggest that the SUMO-2/3 conjugation a of acute and The of SUMO-2/3 conjugation and highly of the regulation of the SUMO-2/3 conjugation in the of environmental stress. of the SUMO-2/3 conjugation in to environmental not the SUMO-2/3 conjugation but the regulation of the SUMO-2/3 environmental the SUMO-1 and modification pathways be by modification of is a major SUMO-1 substrate, but is by to there is of modification of cells and SUMO-1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar). of support the of modification of by SUMO-1 in have that is by SUMO-2 and -3 as as by SUMO-1 when SUMO-2 -3 is in cell T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we identified the modification of by SUMO-1 not in but in cells such as human and and and a be that of SUMO-2 -3 by the of the modification that the of modification between SUMO-1 and is there be two in cells that the modification of proteins the SUMO-1 there is for the of two of human 1996; Google and of 1998; PubMed Scopus Google Scholar). is an has been demonstrated to be for SUMO-1 conjugation to H. T. Pu R.T. T. Dasso M. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Melchior F. M.J. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). be to these in of for SUMO-1 to the conjugation the of a isopeptide that and the of SUMO-1 conjugation to RanGAP1. for this is as has been shown to and H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, H. Pu R. M. Dasso M. Sci. 1997; PubMed Scopus Google Scholar). SUMO-1 and be conjugated to by of SUMO-2/3 RanGAP1. This in the of SUMO-1 modification of RanGAP1. The and that 1998; PubMed Scopus Google Scholar, T. H. T. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the the (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google for the SUMO-2/3 but SUMO-2/3 has not been of SUMO-2/3 revealed multiple in the of of these proteins were in the that a of cellular proteins by SUMO-2/3 as to SUMO-1. that of the high molecular mass were in to the high molecular mass that in the the of these proteins remain to be for SUMO-1 and conjugation including SUMO-1 and This for the of of the SUMO-1 and modification at to detected by antibody have been the nuclear large complex known as nuclear domain 10 nuclear protein M.J. EMBO J. 1998; PubMed Scopus Google Scholar, T. H. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Freemont H. Dasso M. M. J. J. 1998; PubMed Google Scholar, T. Yeh J. Cell Biol. PubMed Scopus Google Scholar). The functions of not but and to be the cell cycle and to to environmental and infection of We found that the nuclear detected by antibody to in SUMO-1 we that has been to the of M.J. EMBO J. 1998; PubMed Scopus Google induced the of SUMO-1 and in the nuclear not results suggest that SUMO-1 and of and of the SUMO-2/3 in the of that there is between the SUMO-1 and is be in the SUMO-2 be important in the to a antibody that between SUMO-2 and -3 but this is to be by the high between SUMO-2 and we have to SUMO-2/3 and have found that a of proteins in The of this antibody and antibody revealed between the SUMO-1 and modification SUMO-2/3 is in greater than and there is a large pool of non-conjugated SUMO-2/3 in cells. this pool of SUMO-2/3 was and high molecular mass proteins by protein-damaging such as is by but by SUMO-2/3 in and to at the than the SUMO-2/3-modified that be if these results that and is and -3 modification and suggest a role for SUMO-2/3 in the response to environmental stress. SUMO-1 is a highly conserved, smallubiquitin-related modifier that has been shown to be covalently conjugated to a variety of cellular proteins (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar). Like ubiquitin, SUMO-1 is believed to form an isopeptide bond between the carboxyl terminus of SUMO-1 and a lysine side chain(s) of the target protein (2.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 15: 5509-5519Crossref Scopus (440) Google Scholar, 3.Kamitani T. Nguyen H.P. Yeh E.T. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 5.Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar). The conjugation of SUMO-1 to cellular proteins has been implicated in multiple vital cellular processes, including nuclear transport, cell cycle control, oncogenesis, inflammation, and the response to virus infection (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 7.Saitoh H. Pu R.T. Dasso M. Trends Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 8.Hodges M. Tissot C. Freemont Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). has been that SUMO-1 conjugation to conjugation R.T. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google that SUMO-1 conjugation the target cellular R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google Scholar, M.J. EMBO J. 1998; PubMed Scopus Google Scholar). known SUMO-1 in vertebrate the Ran GTPase-activating protein is the and is a highly protein that a nuclear protein for H. I. H. Sci. PubMed Scopus Google Scholar, F. Gerace L. Trends Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). has been demonstrated in and in that a lysine at in the terminus of is by SUMO-1 R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google Scholar). large of to be the nuclear a of in the nuclear pore complex (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google Scholar, 4.Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, H. Pu R. M. Dasso M. Sci. 1997; PubMed Scopus Google Scholar). is in the that the conjugation of a ubiquitin-like at the domain target to the R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, M.J. J. Blobel G. J. Cell Biol. 1998; 140: PubMed Scopus Google Scholar). cDNAs for proteins related to SUMO-1 have been human and and to as SUMO-2/SMT3A and (6.Johnson P.R. Hochstrasser M. Trends Cell Biol. 1997; 7: 408-413Abstract Full Text PDF PubMed Scopus (69) Google Scholar, F. C. 1997; PubMed Scopus Google Scholar, H. Biochem. Biol. 1998; Google Scholar). in a wide of and cell that a role in cellular processes. of the of and -3 has revealed that SUMO-1 SUMO-2 and SUMO-2 and -3 is to SUMO-2 and -3 a SUMO-1. has been that SUMO-2 and -3 be to proteins in a to that of SUMO-1 when SUMO-2/3 was in cells T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This that SUMO-2/3 in a to that of SUMO-1. is the of SUMO-2/3 and that support cellular distinctions between two a of the role of SUMO-2/3 and the regulation of SUMO-2/3 SUMO-1 conjugation in vertebrate we have to SUMO-2/3 at the protein in We describe herein the of an antibody to SUMO-2 and this antibody and a we found that was by but by a of SUMO-2/3 than SUMO-1 in We demonstrated that the SUMO-2/3 conjugation be by protein-damaging This is the for the of a ubiquitin-like protein modification in cellular these we that there is regulation of SUMO-2/3 modification as SUMO-1 modification and that the SUMO-2/3 constitute an of the cellular response to environmental stress. the of the known smallubiquitin-related be two in including human SUMO-1 and and the human and in for proteins to SUMO-2/3 revealed that SUMO-2/3 is highly not in vertebrate but in a role for the SUMO-2/3 conjugation a we demonstrated that and proteins in vertebrate cells. between SUMO-1 and was the of a large pool of non-conjugated SUMO-2/3 in cells. This of SUMO-2/3 to be for conjugation conjugation be induced by cellular such as acute and addition, known to the of proteins in the cells and to J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Cell. Biol. PubMed Scopus Google Scholar). Recently, we found that by the has been to protein Trends Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google the conjugation of SUMO-2/3 to a high molecular mass protein not We in the of proteins by SUMO-2/3 in a and the of such SUMO-2/3-modified proteins in has been that the to a wide variety of protein-damaging and environmental 15: PubMed Scopus Google Scholar). Intriguingly, M. J. Cell Biol. PubMed Scopus Google Scholar, EMBO J. PubMed Scopus Google and F. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google have been known to the quantitative of to the conjugated of the SUMO-2/3 conjugation in this that conjugation to cellular proteins of the by the of response in the SUMO-2/3 a by proteins identified and SUMO-2/3 conjugation the target protein by conjugation and the suggest that an for the be conjugated SUMO-1 R.T. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). conjugated SUMO-1 but subjected to of and is that these SUMO-2/3 conjugation the and the of such protein by by SUMO-1 has not been to protein results suggest that the SUMO-2/3 conjugation a of acute and The of SUMO-2/3 conjugation and highly of the regulation of the SUMO-2/3 conjugation in the of environmental stress. of the SUMO-2/3 conjugation in to environmental not the SUMO-2/3 conjugation but the regulation of the SUMO-2/3 environmental the SUMO-1 and modification pathways be by modification of is a major SUMO-1 substrate, but is by to there is of modification of cells and SUMO-1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar). of support the of modification of by SUMO-1 in have that is by SUMO-2 and -3 as as by SUMO-1 when SUMO-2 -3 is in cell T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we identified the modification of by SUMO-1 not in but in cells such as human and and and a be that of SUMO-2 -3 by the of the modification that the of modification between SUMO-1 and is there be two in cells that the modification of proteins the SUMO-1 there is for the of two of human 1996; Google and of 1998; PubMed Scopus Google Scholar). is an has been demonstrated to be for SUMO-1 conjugation to H. T. Pu R.T. T. Dasso M. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Melchior F. M.J. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). be to these in of for SUMO-1 to the conjugation the of a isopeptide that and the of SUMO-1 conjugation to RanGAP1. for this is as has been shown to and H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, H. Pu R. M. Dasso M. Sci. 1997; PubMed Scopus Google Scholar). SUMO-1 and be conjugated to by of SUMO-2/3 RanGAP1. This in the of SUMO-1 modification of RanGAP1. The and that 1998; PubMed Scopus Google Scholar, T. H. T. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the the (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google for the SUMO-2/3 but SUMO-2/3 has not been of SUMO-2/3 revealed multiple in the of of these proteins were in the that a of cellular proteins by SUMO-2/3 as to SUMO-1. that of the high molecular mass were in to the high molecular mass that in the the of these proteins remain to be for SUMO-1 and conjugation including SUMO-1 and This for the of of the SUMO-1 and modification at to detected by antibody have been the nuclear large complex known as nuclear domain 10 nuclear protein M.J. EMBO J. 1998; PubMed Scopus Google Scholar, T. H. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Freemont H. Dasso M. M. J. J. 1998; PubMed Google Scholar, T. Yeh J. Cell Biol. PubMed Scopus Google Scholar). The functions of not but and to be the cell cycle and to to environmental and infection of We found that the nuclear detected by antibody to in SUMO-1 we that has been to the of M.J. EMBO J. 1998; PubMed Scopus Google induced the of SUMO-1 and in the nuclear not results suggest that SUMO-1 and of and of the SUMO-2/3 in the of that there is between the SUMO-1 and is be in the SUMO-2 be important in the to a antibody that between SUMO-2 and -3 but this is to be by the high between SUMO-2 and we have to SUMO-2/3 and have found that a of proteins in The of this antibody and antibody revealed between the SUMO-1 and modification SUMO-2/3 is in greater than and there is a large pool of non-conjugated SUMO-2/3 in cells. this pool of SUMO-2/3 was and high molecular mass proteins by protein-damaging such as is by but by SUMO-2/3 in and to at the than the SUMO-2/3-modified that be if these results that and is and -3 modification and suggest a role for SUMO-2/3 in the response to environmental stress. the of the known smallubiquitin-related be two in including human SUMO-1 and and the human and in for proteins to SUMO-2/3 revealed that SUMO-2/3 is highly not in vertebrate but in a role for the SUMO-2/3 conjugation a we demonstrated that and proteins in vertebrate cells. between SUMO-1 and was the of a large pool of non-conjugated SUMO-2/3 in cells. This of SUMO-2/3 to be for conjugation conjugation be induced by cellular such as acute and addition, known to the of proteins in the cells and to J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Cell. Biol. PubMed Scopus Google Scholar). Recently, we found that by the has been to protein Trends Cell Biol. 1998; Full Text Full Text PDF PubMed Scopus Google the conjugation of SUMO-2/3 to a high molecular mass protein not We in the of proteins by SUMO-2/3 in a and the of such SUMO-2/3-modified proteins in cells. has been that the to a wide variety of protein-damaging and environmental 15: PubMed Scopus Google Scholar). Intriguingly, M. J. Cell Biol. PubMed Scopus Google Scholar, EMBO J. PubMed Scopus Google and F. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google have been known to the quantitative of to the conjugated of the SUMO-2/3 conjugation in this that conjugation to cellular proteins of the by the of response in the SUMO-2/3 a by proteins identified and SUMO-2/3 conjugation the target protein by conjugation and the suggest that an for the be conjugated SUMO-1 R.T. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). conjugated SUMO-1 but subjected to of and is that these SUMO-2/3 conjugation the and the of such protein by by SUMO-1 has not been to protein results suggest that the SUMO-2/3 conjugation a of acute and The of SUMO-2/3 conjugation and highly of the regulation of the SUMO-2/3 conjugation in the of environmental stress. of the SUMO-2/3 conjugation in to environmental not the SUMO-2/3 conjugation but the regulation of the SUMO-2/3 environmental stress. the SUMO-1 and modification pathways be by modification of is a major SUMO-1 substrate, but is by to there is of modification of cells and SUMO-1 (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (236) Google Scholar, H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar). of support the of modification of by SUMO-1 in have that is by SUMO-2 and -3 as as by SUMO-1 when SUMO-2 -3 is in cell T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Nguyen H.P. T. Yeh E.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we identified the modification of by SUMO-1 not in but in cells such as human and and and a be that of SUMO-2 -3 by the of the modification The that the of modification between SUMO-1 and is there be two in cells that the modification of proteins the SUMO-1 there is for the of two of human 1996; Google and of 1998; PubMed Scopus Google Scholar). is an has been demonstrated to be for SUMO-1 conjugation to H. T. Pu R.T. T. Dasso M. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Melchior F. M.J. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). be to these in of for SUMO-1 to the conjugation the of a isopeptide that and the of SUMO-1 conjugation to RanGAP1. for this is as has been shown to and H. Dasso M. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar, H. Pu R. M. Dasso M. Sci. 1997; PubMed Scopus Google Scholar). SUMO-1 and be conjugated to by of SUMO-2/3 RanGAP1. This in the of SUMO-1 modification of RanGAP1. The and that 1998; PubMed Scopus Google Scholar, T. H. T. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the the (1.Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (949) Google for the SUMO-2/3 but SUMO-2/3 has not been of SUMO-2/3 revealed multiple in the of of these proteins were in the that a of cellular proteins by SUMO-2/3 as to SUMO-1. that of the high molecular mass were in to the high molecular mass that in the the of these proteins remain to be for SUMO-1 and conjugation including SUMO-1 and This for the of of the SUMO-1 and modification at to detected by antibody have been the nuclear large complex known as nuclear domain 10 nuclear protein M.J. EMBO J. 1998; PubMed Scopus Google Scholar, T. H. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Freemont H. Dasso M. M. J. J. 1998; PubMed Google Scholar, T. Yeh J. Cell Biol. PubMed Scopus Google Scholar). The functions of not but and to be the cell cycle and to to environmental and infection of We found that the nuclear detected by antibody to in SUMO-1 we that has been to the of M.J. 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The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8 + T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Gurin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (ΔsecA2) with a strongly attenuating lysine auxotrophy mutation (ΔlysA). The ΔsecA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8(+) T cells in vivo. Similarly, the ΔsecA2ΔlysA strain retained enhanced apoptosis and augmented CD8(+) T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ΔsecA2ΔlysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.

Fragile X is the most common monogenic disorder associated with intellectual disability (ID) and autism spectrum disorders (ASD). Additionally, many patients are afflicted with executive dysfunction, ADHD, seizure disorder and sleep disturbances. Fragile X is caused by loss of FMRP expression, which is encoded by the FMR1 gene. Both the fly and mouse models of fragile X are also based on having no functional protein expression of their respective FMR1 homologs. The fly model displays well defined cognitive impairments and structural brain defects and the mouse model, although having subtle behavioral defects, has robust electrophysiological phenotypes and provides a tool to do extensive biochemical analysis of select brain regions. Decreased cAMP signaling has been observed in samples from the fly and mouse models of fragile X as well as in samples derived from human patients. Indeed, we have previously demonstrated that strategies that increase cAMP signaling can rescue short term memory in the fly model and restore DHPG induced mGluR mediated long term depression (LTD) in the hippocampus to proper levels in the mouse model (McBride et al., 2005; Choi et al., 2011, 2015). Here, we demonstrate that the same three strategies used previously with the potential to be used clinically, lithium treatment, PDE-4 inhibitor treatment or mGluR antagonist treatment can rescue long term memory in the fly model and alter the cAMP signaling pathway in the hippocampus of the mouse model.