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BMC Cell Biology Biomed Central BMC Cell Biology BioMed Central Research article Open Access Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein Xiaomin Zhang*1, Gohar Azhar3, Scott Helms1, Ying Zhong1 and Jeanne Y Wei*1,2 Address: 1From the Donald W. Reynolds Department of Geriatrics, The University of Arkansas for Medical Sciences and Geriatric Research, Education, and Clinical Center, Little Rock, AR, USA, 2Central Arkansas Veterans Healthcare System, Geriatric and Extended Care, Little Rock, AR, USA and 3Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Email: Xiaomin Zhang* - [email protected]; Gohar Azhar - [email protected]; Scott Helms - [email protected]; Ying Zhong - [email protected]; Jeanne Y Wei* - [email protected] * Corresponding authors Published: 29 January 2008 Received: 1 August 2007 Accepted: 29 January 2008 BMC Cell Biology 2008, 9:8 doi:10.1186/1471-2121-9-8 This article is available from: http://www.biomedcentral.com/1471-2121/9/8 © 2008 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results: In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion: These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging. Background in the myocardium of human and mouse with advancing A novel protein, p49/STRAP, was recently identified in age, and also in transgenic mice suffering from cardiomy- our laboratory [1]. The initial observations indicated that opathy [1,2]. p49/STRAP is a cofactor of serum response factor (SRF) contributing to the regulation of SRF target genes in the Our previous studies suggested that the expression of a heart. This gene was later named by The Human Genome number of cardiac genes are regulated by a protein com- Organization (HUGO) as SRFBP1 based on our initial plex containing SRF and it's multiple binding proteins, finding that p49/STRAP binds to SRF. The p49/STRAP including p49/STRAP and Zipzap/p200 [1-3]. We had mRNA and protein are expressed in multiple tissues, noticed that the protein complexes obtained by immuno- including heart, brain, liver, skeletal muscle and kidney. precipitation assays with either p49/STRAP or SRF anti- An increase of p49/STRAP expression has been observed body contained more components than we had Page 1 of 11 (page number not for citation purposes) BMC Cell Biology 2008, 9:8 http://www.biomedcentral.com/1471-2121/9/8 anticipated, suggesting that additional proteins may be into the pcDNA3-Flag vector. A pcDNA3-mCherry- present in the complex [1-3]. The p49/STRAP protein NDUFAB1 construct was generated by ligating the interacts directly with the SRF transcriptional activation mCherry red fluorescent protein (RFP, a gift from Dr. domain. Therefore, it is possible that a protein complex Roger Tsien, University of California, San Diego) in frame formed around this domain or those proteins that bind to with NDUFAB1 into the pcDNA3 vector. The p49/STRAP- p49/STRAP may have a significant effect on SRF activa- EGFP fusion protein construct and the pcDNA3-HA-p49/ tion. In an attempt to determine the identity of these puta- STRAP construct were generated as previously reported tive proteins, we used p49/STRAP protein fragments as [1]. All the plasmid constructs were subjected to sequenc- bait in the yeast two-hybrid system. We found that ing analysis to verify the open reading frame sequence. NDUFAB1, a subunit of NADH dehydrogenase (a nuclear encoded subunit of mitochondrial Complex I), repre- Generation of Recombinant Adenoviruses sented the majority of cDNA clones interacting with p49/ The p49/STRAP recombinant adenovirus was generated STRAP in the multiple screenings. Overexpression of p49 using the AdEasy system, a generous gift from Dr. Vogel- changed the intracellular NAD/NADH ratio and reduced stein (The Johns Hopkins Oncology Center, Baltimore) the amount of acetylated-SRF protein. Therefore, p49 may [12]. The generation, purification and titration of the play a role in the process of protein acetylation and recombinant adenoviruses Ad-EGFP and Ad-p49/STRAP deacetylation, thereby modulating transcriptional regula- were performed according to the protocol described by He tion. et al. [12]. Methods Tissue Culture and Transfection Assays Yeast Two-hybrid Screening and Bioinformatic Analysis The H9C2 cells, NIH3T3 cells and neonatal rat cardiac Yeast two-hybrid screening was performed as previously myocytes were cultured as previously described [1,13,14]. described [1]. Briefly, one "bait" construct containing the Transient transfection was carried out using the desired N-terminus (residues 1–256) and another one containing expression plasmid constructs and the Lipofectamine the C-terminus (residues 250–427) of the p49/STRAP 2000 reagent (Invitrogen). At approximately 4 hr after the protein were used to screen cDNA libraries generated from transfection was initiated, cells were placed in DMEM a human heart and Hela cells (Clontech). A total of three medium containing 10% newborn bovine serum and separate screenings was performed. Positive target cDNA incubated at 37°C overnight. clones were subjected to a series of analyses according to the manufacturer's instruction manual. The final positive Co-immunoprecipitation and Western Blotting clones were sequenced and their sequences were com- The expression plasmid constructs containing p49/STRAP pared with the Genbank nucleotide database. and NDUFAB1 were cotransfected into NIH3T3 cells by using Lipofectamine 2000 (Invitrogen) as previously Bioinformatics Analysis described [1]. At 48 h after the transfection, cells were har- The p49/STRAP and NDUFAB1 sequences were subjected vested, and the whole-cell lysate was isolated. The co- to analyses using web-based and/or freely-downloaded immunoprecipitation and Western blotting were carried bioinformatic tools, including "PSORT II" [4], "iPSORT" out as previously described [1]. Antibodies that were [5], "WoLF PSORT", "BaCelLo" [6], "pTARGET" [7], employed included monoclonal antibodies HA.11 (Cov- "CELLO version 2" [8], "Golgi Localization Predictor" [9], ance), FLAG (M2, Sigma), SRF (Abcam) and an anti- "HSLPred" [10], "GoCore" and "SignalP" [11]. acetyl-Lysine antibody (clone 4G12, Upsate); polyclonal antibodies HA (Santa Cruz), Flag (Sigma), a SRF antibody Cloning of Full-length Coding Region Sequence of which recognizes the C-terminus of SRF protein (Santa NDUFAB1 gene Cruz) and p49/Strap [1]. The majority of the cDNA clones interacting with the N- terminal bait of the p49/STRAP protein in the yeast two- Fluorescence Microscopy hybrid screening matched a single gene in the GenBank The expression plasmids "EGFP-p49/STRAP" and database, NDUFAB1, which is a subunit of NADH dehy- "mCherry-NDUFAB1" were used for the transfection. At drogenase (OMIM 25010; EC 1.6.5.3). The full-length approximately 24, 48, and 72 hours after transfection, the coding region of the human NDUFAB1 gene was ampli- expression of EGFP-p49/STRAP and mCherry-NDUFAB1 fied by PCR using a human heart cDNA sample (Clon- were examined by fluorescence microscopy using a Nikon tech) and then subcloned into the expression vectors. Eclipse E600 equipped with CoolSNAP-ES digital camera and MetaVue 6.2 software. Plasmid Constructs A pcDNA3-Flag-NDUFAB1 construct was generated by The intensity of fluorescence was measured using the ligating the NDUFAB1 coding region in frame with Flag "ImageJ 1.37" program (National Institutes of Health, Page 2 of 11 (page number not for citation purposes) BMC Cell Biology 2008, 9:8 http://www.biomedcentral.com/1471-2121/9/8 Bethesda, MD). Background noise was subtracted. The proteins that may selectively interact with the different area of, and total fluorescence signal from, nuclear and parts of the p49 protein. non-nuclear cellular regions were measured separately. The cytoplasm superior and inferior to the nucleus was A human heart cDNA library derived from terminally dif- estimated to have a p49/STRAP concentration similar to ferentiated cardiomyocytes and non-cardiomyocyte cells, that in the perinuclear region. It was further estimated that and a Hela cell cDNA library generated from highly prolif- the cytoplasmic overlay on the nucleus was thinner than erating tumor cells were selected to represent the cells that of the surrounding region. For these reasons, the total with different biological features. fluorescence in the nuclear region was estimated to con- sist of p49 in the nucleus
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