- Haas, Peter (x)
- 2008-08-01 (x)
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Show moreBetaine homocysteine methyltransferase-2 (BHMT-2) is a zinc metalloenzyme that plays an important role in the regulation of the homocysteine metabolism. It uses S-methylmethionine (SMM) as a methyl donor for the methylation of homocysteine. BHMT-2 is a 73% homologous to the protein BHMT, which catalyzes the remethylation of betaine and homocysteine to form dimethylglycine and methionine. BHMT is abundant in the human liver, kidney, and lens of the eye. BHMT (and likely BHMT-2) are involved in regulating homocysteine and S-adenosyl-L-methionine (SAM, AdoMet) levels. The goal of this study was to generate recombinant BHMT-2 for structural and functional studies. Escherichia coli BL21 (DE3), were transformed with the expression construct coded for BHMT2, fused with a N- terminal His-tag, and then were grown. Recombinant BHTM-2 protein was purified using 6xHis-tag affinity chromatography. Also, size exclusion chromatography was used to purify His-BHMT-2. The dimer form of His-BHMT-2 protein was obtained as a result in the study. By the analyzed results of this study, the protein BHMT-2 can be purified by different methods of chromatography. BHMT-2 polymorphisms may contribute to BHMT-2 enzyme variants with different levels of activity, thus leading to different levels of SAM (aka AdoMet), which influences acetaminophen sensitivity/response and thus liver toxicity. However, further experiments are needed to make structural studies (crystallography) and functional (enzyme assays) to compare the WT and variant enzymes (corresponding to genetic polymorphisms in the gene). It is very important to make further studies in way to determine the mechanism and function of this protein in humans.
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Show moreActivating mutations in SHP-2 tyrosine phosphatase implicated in cell signaling processes have been identified in various childhood leukemias and sporadic solid tumors. However, the molecular mechanisms by which the activating mutations in SHP-2 induce malignancies are not fully understood. In this study, we investigated potential involvement of SHP-2 in chromosome segregation during mitosis, dysregulation of which leads to genome instability and malignancies. We determined localization of SHP-2 in the kinetochore, a crucial protein complex structure connecting microtubules and chromosomes. We found that SHP-2 localized at the kinetochore. Our findings suggest that SHP-2 may be involved in chromosome segregation and can help to answer how SHP-2 mutations can induce genetic instability and thereby malignancies.
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Show moreCited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D) rich C-terminaldomain) is a member of the transcription factor modulators family. Cited2 is essential for normal development in mice. Mice with a total knockout (KO) of this gene died within the first 2-3 weeks of development and presented malformations in liver, eye lens, and heart. In the liver Cited2 is known to interact with HNF4-alpha to mediate functions essential for liver development, differentiation and functions. Our main focus is to identify the role of Cited2 in the adult liver by performing in vivo and in vitro experiments. In our in vivo experiment, Cited2 liver specific KO mice were generated followed by analyzing their blood biochemistry and glucose tolerance. Liver specific KO mice and wild type mice did not present differences in their blood biochemistry and their body weight. In contrast, a difference in glucose tolerance was observed between KO and wild type mice. In the in vitro experiment, the effect of Cited2 expression on cell proliferation was studied in a human liver cell line, Sk-Hep1. These cells were treated with butyrate (a HDAC inhibitor) and viability was measured by the MTT assay and Cited2 mRNA expression was analyzed using Real Time PCR. The data showed that butyrate (HDAC inhibitor) affects the expression of Cited2 and cell proliferation. Taken together, these results suggest Cited2 may play a role in liver functions in adult mice. More detailed experiments will have to be pursued in order to find specific pathways involved.
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