Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase
Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is ex...
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creator | Lai, T. S Hausladen, A Slaughter, T. F Eu, J. P Stamler, J. S Greenberg, C. S |
description | Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca2+, up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca2+ to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity. |
doi_str_mv | 10.1021/bi002321t |
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S ; Hausladen, A ; Slaughter, T. F ; Eu, J. P ; Stamler, J. S ; Greenberg, C. S</creator><creatorcontrib>Lai, T. S ; Hausladen, A ; Slaughter, T. F ; Eu, J. P ; Stamler, J. S ; Greenberg, C. S</creatorcontrib><description>Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca2+, up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca2+ to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi002321t</identifier><identifier>PMID: 11305905</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Adenosine Diphosphate - physiology ; Adenosine Triphosphate - pharmacology ; Animals ; Calcium - physiology ; Cations, Divalent - pharmacology ; Cattle ; Cells, Cultured ; Cysteine - analogs & derivatives ; Cysteine - pharmacology ; Endothelium, Vascular - enzymology ; Endothelium, Vascular - metabolism ; Enzyme Activation - drug effects ; Enzyme Inhibitors - pharmacology ; GTP-Binding Proteins - antagonists & inhibitors ; GTP-Binding Proteins - chemistry ; GTP-Binding Proteins - genetics ; GTP-Binding Proteins - metabolism ; Guanosine Triphosphate - pharmacology ; Guinea Pigs ; Humans ; Kinetics ; Mercaptoethanol ; Nitric Oxide - metabolism ; Nitroso Compounds - metabolism ; Nitroso Compounds - pharmacology ; Phosphorylcholine - analogs & derivatives ; Phosphorylcholine - metabolism ; Platelet Aggregation ; Protein Conformation ; Recombinant Proteins - metabolism ; S-Nitrosothiols ; Sphingosine - analogs & derivatives ; Sphingosine - metabolism ; Transglutaminases - antagonists & inhibitors ; Transglutaminases - chemistry ; Transglutaminases - genetics ; Transglutaminases - metabolism</subject><ispartof>Biochemistry (Easton), 2001-04, Vol.40 (16), p.4904-4910</ispartof><rights>Copyright © 2001 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a349t-81f959fc00e2d0f2cbc61d50b2d240c7ff9ae9cc67beb691b4aabdca596a20243</citedby><cites>FETCH-LOGICAL-a349t-81f959fc00e2d0f2cbc61d50b2d240c7ff9ae9cc67beb691b4aabdca596a20243</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi002321t$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi002321t$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11305905$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lai, T. S</creatorcontrib><creatorcontrib>Hausladen, A</creatorcontrib><creatorcontrib>Slaughter, T. F</creatorcontrib><creatorcontrib>Eu, J. P</creatorcontrib><creatorcontrib>Stamler, J. S</creatorcontrib><creatorcontrib>Greenberg, C. S</creatorcontrib><title>Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca2+, up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca2+ to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.</description><subject>Adenosine Diphosphate - physiology</subject><subject>Adenosine Triphosphate - pharmacology</subject><subject>Animals</subject><subject>Calcium - physiology</subject><subject>Cations, Divalent - pharmacology</subject><subject>Cattle</subject><subject>Cells, Cultured</subject><subject>Cysteine - analogs & derivatives</subject><subject>Cysteine - pharmacology</subject><subject>Endothelium, Vascular - enzymology</subject><subject>Endothelium, Vascular - metabolism</subject><subject>Enzyme Activation - drug effects</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>GTP-Binding Proteins - antagonists & inhibitors</subject><subject>GTP-Binding Proteins - chemistry</subject><subject>GTP-Binding Proteins - genetics</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>Guanosine Triphosphate - pharmacology</subject><subject>Guinea Pigs</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Mercaptoethanol</subject><subject>Nitric Oxide - metabolism</subject><subject>Nitroso Compounds - metabolism</subject><subject>Nitroso Compounds - pharmacology</subject><subject>Phosphorylcholine - analogs & derivatives</subject><subject>Phosphorylcholine - metabolism</subject><subject>Platelet Aggregation</subject><subject>Protein Conformation</subject><subject>Recombinant Proteins - metabolism</subject><subject>S-Nitrosothiols</subject><subject>Sphingosine - analogs & derivatives</subject><subject>Sphingosine - metabolism</subject><subject>Transglutaminases - antagonists & inhibitors</subject><subject>Transglutaminases - chemistry</subject><subject>Transglutaminases - genetics</subject><subject>Transglutaminases - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkFtLAzEQhYMotlYf_AOyLwqCq5PsrXks9Qr1Qrv64kOYzWZLdC91kxX77420VASfhjPzcYZzCDmkcE6B0YtMA7CAUbtF-jRi4IecR9ukDwCxz3gMPbJnzJuTISThLulRGkDEIeqT1zGWUneVN1XzrkSrjDfzH7RtG7N0Ujf1mXep6r8LrHNvJK3-1HbpNYWXamM65aUt1mZedhYrXaNR-2SnwNKog_UckOfrq3R8608eb-7Go4mPQcitP6QFj3ghARTLoWAykzHNI8hYzkKQSVFwVFzKOMlUFnOahYhZLjHiMTJgYTAgJyvfRdt8dMpYUWkjVVlirZrOiCRxYYMhOPB0BUqXxrSqEItWV9guBQXx06TYNOnYo7Vpl1Uq_yXX1TnAXwHaWPW1uWP7LuIkSCKRPs3EfTIJQhhOxYvjj1c8SiPemq6tXSf_PP4G8jmK3A</recordid><startdate>20010424</startdate><enddate>20010424</enddate><creator>Lai, T. S</creator><creator>Hausladen, A</creator><creator>Slaughter, T. F</creator><creator>Eu, J. P</creator><creator>Stamler, J. S</creator><creator>Greenberg, C. S</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20010424</creationdate><title>Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase</title><author>Lai, T. S ; Hausladen, A ; Slaughter, T. F ; Eu, J. P ; Stamler, J. S ; Greenberg, C. S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a349t-81f959fc00e2d0f2cbc61d50b2d240c7ff9ae9cc67beb691b4aabdca596a20243</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Adenosine Diphosphate - physiology</topic><topic>Adenosine Triphosphate - pharmacology</topic><topic>Animals</topic><topic>Calcium - physiology</topic><topic>Cations, Divalent - pharmacology</topic><topic>Cattle</topic><topic>Cells, Cultured</topic><topic>Cysteine - analogs & derivatives</topic><topic>Cysteine - pharmacology</topic><topic>Endothelium, Vascular - enzymology</topic><topic>Endothelium, Vascular - metabolism</topic><topic>Enzyme Activation - drug effects</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>GTP-Binding Proteins - antagonists & inhibitors</topic><topic>GTP-Binding Proteins - chemistry</topic><topic>GTP-Binding Proteins - genetics</topic><topic>GTP-Binding Proteins - metabolism</topic><topic>Guanosine Triphosphate - pharmacology</topic><topic>Guinea Pigs</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Mercaptoethanol</topic><topic>Nitric Oxide - metabolism</topic><topic>Nitroso Compounds - metabolism</topic><topic>Nitroso Compounds - pharmacology</topic><topic>Phosphorylcholine - analogs & derivatives</topic><topic>Phosphorylcholine - metabolism</topic><topic>Platelet Aggregation</topic><topic>Protein Conformation</topic><topic>Recombinant Proteins - metabolism</topic><topic>S-Nitrosothiols</topic><topic>Sphingosine - analogs & derivatives</topic><topic>Sphingosine - metabolism</topic><topic>Transglutaminases - antagonists & inhibitors</topic><topic>Transglutaminases - chemistry</topic><topic>Transglutaminases - genetics</topic><topic>Transglutaminases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lai, T. S</creatorcontrib><creatorcontrib>Hausladen, A</creatorcontrib><creatorcontrib>Slaughter, T. F</creatorcontrib><creatorcontrib>Eu, J. P</creatorcontrib><creatorcontrib>Stamler, J. S</creatorcontrib><creatorcontrib>Greenberg, C. S</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lai, T. S</au><au>Hausladen, A</au><au>Slaughter, T. F</au><au>Eu, J. P</au><au>Stamler, J. S</au><au>Greenberg, C. S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2001-04-24</date><risdate>2001</risdate><volume>40</volume><issue>16</issue><spage>4904</spage><epage>4910</epage><pages>4904-4910</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca2+, up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca2+ to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>11305905</pmid><doi>10.1021/bi002321t</doi><tpages>7</tpages></addata></record> |
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subjects | Adenosine Diphosphate - physiology Adenosine Triphosphate - pharmacology Animals Calcium - physiology Cations, Divalent - pharmacology Cattle Cells, Cultured Cysteine - analogs & derivatives Cysteine - pharmacology Endothelium, Vascular - enzymology Endothelium, Vascular - metabolism Enzyme Activation - drug effects Enzyme Inhibitors - pharmacology GTP-Binding Proteins - antagonists & inhibitors GTP-Binding Proteins - chemistry GTP-Binding Proteins - genetics GTP-Binding Proteins - metabolism Guanosine Triphosphate - pharmacology Guinea Pigs Humans Kinetics Mercaptoethanol Nitric Oxide - metabolism Nitroso Compounds - metabolism Nitroso Compounds - pharmacology Phosphorylcholine - analogs & derivatives Phosphorylcholine - metabolism Platelet Aggregation Protein Conformation Recombinant Proteins - metabolism S-Nitrosothiols Sphingosine - analogs & derivatives Sphingosine - metabolism Transglutaminases - antagonists & inhibitors Transglutaminases - chemistry Transglutaminases - genetics Transglutaminases - metabolism |
title | Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase |
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