Post by Max on Jul 17, 2005 16:55:54 GMT -5
High circulating concentrations of IL-1ra and IL-6 are independent correlates of hyperhomocysteinemia and may explain, at least in part, the association between homocysteine and atherosclerosis [1].
References:
[1] Gori AM, Corsi AM, Fedi S, Gazzini A, Sofi F, Bartali B, Bandinelli S, Gensini GF, Abbate R, Ferrucci L A proinflammatory state is associated with hyperhomocysteinemia in the elderly. (2005) Am J Clin Nutr. Aug;82(2):335-41.
J Biol Chem. 2001 Nov 23;276(47):43570-9. Epub 2001 Sep 18. Related Articles, Links
Transcriptional regulation of cell-specific expression of the human cystathionine beta -synthase gene by differential binding of Sp1/Sp3 to the -1b promoter.
Ge Y, Matherly LH, Taub JW.
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-3792 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involved in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Matherly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, the critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which differ approximately 10-fold in levels of CBS transcripts transcribed from the CBS -1b promoter. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcriptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger 1 elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared with HepG2 cells despite comparable levels of nuclear factor Y and USF-1 binding and similar levels of Sp1 and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp factor binding in gel shift assays, thus excluding a role for an unknown activator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Increased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding resulted from treatment of HepG2 nuclear extracts with calf alkaline phosphatase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcription factor binding and transactivation of the CBS -1b promoter. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down syndrome.
PMID: 11562358 [PubMed - indexed for MEDLINE]
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0504786102
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Articles by Yamada, K.
Articles by Matthews, R. G.
Biochemistry
Regulation of human methylenetetrahydrofolate reductase by phosphorylation
( homocysteine | posttranslational modification )
Kazuhiro Yamada *, John R. Strahler , Philip C. Andrews , and Rowena G. Matthews *
*Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216; and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-1066
Contributed by Rowena G. Matthews, June 9, 2005
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor for the conversion of homocysteine to methionine. Regulation of MTHFR activity is crucial for maintaining cellular concentrations of methionine and S-adenosylmethionine (AdoMet). Purified recombinant human MTHFR expressed in insect cells is multiply phosphorylated on an N-terminal extension of the protein that contains a highly conserved serine-rich region. Treatment by alkaline phosphatase removes seven phosphoryl groups from the enzyme. Thr-34 was identified as one of the seven phosphorylation sites by using a monoclonal antibody directed toward pThr-Pro. Mutation of Thr-34 to Ala completely blocks modification as judged by mass spectrometric analysis, suggesting that Thr-34 is the priming phosphorylation site. The Thr34Ala mutant was expressed in baculovirus-infected insect cells, and its enzymic properties were compared with wild-type enzyme. The mutant enzyme and alkaline phosphatase-treated wild-type enzyme are more active than untreated wild-type enzyme and less sensitive to inhibition by saturating AdoMet, indicating that phosphorylation at Thr-34 is critical for allosteric regulation of human MTHFR activity by AdoMet. The absence of methionine and the presence of adenosine in the cell culture medium, which lead to a low intracellular AdoMet/S-adenosylhomocysteine ratio, are associated with faster electrophoretic mobility of MTHFR, presumably because of less or no phosphorylation. Because the faster-mobility MTHFR is associated with the more active form of MTHFR, this response is likely to increase methionine production. Those observations suggest that AdoMet functions not only as an allosteric inhibitor but also to control phosphorylation of human MTHFR.
References:
[1] Gori AM, Corsi AM, Fedi S, Gazzini A, Sofi F, Bartali B, Bandinelli S, Gensini GF, Abbate R, Ferrucci L A proinflammatory state is associated with hyperhomocysteinemia in the elderly. (2005) Am J Clin Nutr. Aug;82(2):335-41.
J Biol Chem. 2001 Nov 23;276(47):43570-9. Epub 2001 Sep 18. Related Articles, Links
Transcriptional regulation of cell-specific expression of the human cystathionine beta -synthase gene by differential binding of Sp1/Sp3 to the -1b promoter.
Ge Y, Matherly LH, Taub JW.
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-3792 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involved in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Matherly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, the critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which differ approximately 10-fold in levels of CBS transcripts transcribed from the CBS -1b promoter. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcriptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger 1 elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared with HepG2 cells despite comparable levels of nuclear factor Y and USF-1 binding and similar levels of Sp1 and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp factor binding in gel shift assays, thus excluding a role for an unknown activator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Increased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding resulted from treatment of HepG2 nuclear extracts with calf alkaline phosphatase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcription factor binding and transactivation of the CBS -1b promoter. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down syndrome.
PMID: 11562358 [PubMed - indexed for MEDLINE]
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0504786102
This Article
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My File Cabinet
Download to citation manager
Request Copyright Permission
PubMed
PubMed Citation
Articles by Yamada, K.
Articles by Matthews, R. G.
Biochemistry
Regulation of human methylenetetrahydrofolate reductase by phosphorylation
( homocysteine | posttranslational modification )
Kazuhiro Yamada *, John R. Strahler , Philip C. Andrews , and Rowena G. Matthews *
*Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216; and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-1066
Contributed by Rowena G. Matthews, June 9, 2005
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, the methyl donor for the conversion of homocysteine to methionine. Regulation of MTHFR activity is crucial for maintaining cellular concentrations of methionine and S-adenosylmethionine (AdoMet). Purified recombinant human MTHFR expressed in insect cells is multiply phosphorylated on an N-terminal extension of the protein that contains a highly conserved serine-rich region. Treatment by alkaline phosphatase removes seven phosphoryl groups from the enzyme. Thr-34 was identified as one of the seven phosphorylation sites by using a monoclonal antibody directed toward pThr-Pro. Mutation of Thr-34 to Ala completely blocks modification as judged by mass spectrometric analysis, suggesting that Thr-34 is the priming phosphorylation site. The Thr34Ala mutant was expressed in baculovirus-infected insect cells, and its enzymic properties were compared with wild-type enzyme. The mutant enzyme and alkaline phosphatase-treated wild-type enzyme are more active than untreated wild-type enzyme and less sensitive to inhibition by saturating AdoMet, indicating that phosphorylation at Thr-34 is critical for allosteric regulation of human MTHFR activity by AdoMet. The absence of methionine and the presence of adenosine in the cell culture medium, which lead to a low intracellular AdoMet/S-adenosylhomocysteine ratio, are associated with faster electrophoretic mobility of MTHFR, presumably because of less or no phosphorylation. Because the faster-mobility MTHFR is associated with the more active form of MTHFR, this response is likely to increase methionine production. Those observations suggest that AdoMet functions not only as an allosteric inhibitor but also to control phosphorylation of human MTHFR.