Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein
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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.
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.
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.
KeywordsNADH Dehydrogenase H9C2 Cell Recombinant Adenovirus Serum Response Factor Neonatal Cardiac Myocytes
Nicotinamide adenine dinucleotide hydride
Nicotinamide adenine dinucleotide
"SRF-dependent transcription regulation-associated protein" which is a 49-kilodalton protein
"serum response factor binding protein 1" which is a synonym of p49/STRAP
NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8 kDa
Serum response factor
"Zinc finger protein with activation potential" which is 200-kilodalton protein
Enhanced green fluorescent protein
Multiplicity of infection
Nuclear localization sequence
A novel protein, p49/STRAP, was recently identified in our laboratory . The initial observations indicated that p49/STRAP is a cofactor of serum response factor (SRF) contributing to the regulation of SRF target genes in the heart. This gene was later named by The Human Genome Organization (HUGO) as SRFBP1 based on our initial finding that p49/STRAP binds to SRF. The p49/STRAP mRNA and protein are expressed in multiple tissues, including heart, brain, liver, skeletal muscle and kidney. An increase of p49/STRAP expression has been observed in the myocardium of human and mouse with advancing age, and also in transgenic mice suffering from cardiomyopathy [1, 2].
Our previous studies suggested that the expression of a number of cardiac genes are regulated by a protein complex containing SRF and it's multiple binding proteins, including p49/STRAP and Zipzap/p200 [1, 2, 3]. We had noticed that the protein complexes obtained by immunoprecipitation assays with either p49/STRAP or SRF antibody contained more components than we had anticipated, suggesting that additional proteins may be present in the complex [1, 2, 3]. The p49/STRAP protein interacts directly with the SRF transcriptional activation domain. Therefore, it is possible that a protein complex formed around this domain or those proteins that bind to p49/STRAP may have a significant effect on SRF activation. In an attempt to determine the identity of these putative proteins, we used p49/STRAP protein fragments as bait in the yeast two-hybrid system. We found that NDUFAB1, a subunit of NADH dehydrogenase (a nuclear encoded subunit of mitochondrial Complex I), represented the majority of cDNA clones interacting with p49/STRAP in the multiple screenings. Overexpression of p49 changed the intracellular NAD/NADH ratio and reduced the amount of acetylated-SRF protein. Therefore, p49 may play a role in the process of protein acetylation and deacetylation, thereby modulating transcriptional regulation.
Yeast Two-hybrid Screening and Bioinformatic Analysis
Yeast two-hybrid screening was performed as previously described . Briefly, one "bait" construct containing the N-terminus (residues 1–256) and another one containing the C-terminus (residues 250–427) of the p49/STRAP protein were used to screen cDNA libraries generated from a human heart and Hela cells (Clontech). A total of three separate screenings was performed. Positive target cDNA clones were subjected to a series of analyses according to the manufacturer's instruction manual. The final positive clones were sequenced and their sequences were compared with the Genbank nucleotide database.
The p49/STRAP and NDUFAB1 sequences were subjected to analyses using web-based and/or freely-downloaded bioinformatic tools, including "PSORT II" , "iPSORT" , "WoLF PSORT", "BaCelLo" , "pTARGET" , "CELLO version 2" , "Golgi Localization Predictor" , "HSLPred" , "GoCore" and "SignalP" .
Cloning of Full-length Coding Region Sequence of NDUFAB1 gene
The majority of the cDNA clones interacting with the N-terminal bait of the p49/STRAP protein in the yeast two-hybrid screening matched a single gene in the GenBank database, NDUFAB1, which is a subunit of NADH dehydrogenase (OMIM 25010; EC 220.127.116.11). The full-length coding region of the human NDUFAB1 gene was amplified by PCR using a human heart cDNA sample (Clontech) and then subcloned into the expression vectors.
A pcDNA3-Flag-NDUFAB1 construct was generated by ligating the NDUFAB1 coding region in frame with Flag into the pcDNA3-Flag vector. A pcDNA3-mCherry-NDUFAB1 construct was generated by ligating the mCherry red fluorescent protein (RFP, a gift from Dr. Roger Tsien, University of California, San Diego) in frame with NDUFAB1 into the pcDNA3 vector. The p49/STRAP-EGFP fusion protein construct and the pcDNA3-HA-p49/STRAP construct were generated as previously reported . All the plasmid constructs were subjected to sequencing analysis to verify the open reading frame sequence.
Generation of Recombinant Adenoviruses
The p49/STRAP recombinant adenovirus was generated using the AdEasy system, a generous gift from Dr. Vogelstein (The Johns Hopkins Oncology Center, Baltimore) . The generation, purification and titration of the recombinant adenoviruses Ad-EGFP and Ad-p49/STRAP were performed according to the protocol described by He et al. .
Tissue Culture and Transfection Assays
The H9C2 cells, NIH3T3 cells and neonatal rat cardiac myocytes were cultured as previously described [1, 13, 14]. Transient transfection was carried out using the desired expression plasmid constructs and the Lipofectamine 2000 reagent (Invitrogen). At approximately 4 hr after the transfection was initiated, cells were placed in DMEM medium containing 10% newborn bovine serum and incubated at 37°C overnight.
Co-immunoprecipitation and Western Blotting
The expression plasmid constructs containing p49/STRAP and NDUFAB1 were cotransfected into NIH3T3 cells by using Lipofectamine 2000 (Invitrogen) as previously described . At 48 h after the transfection, cells were harvested, and the whole-cell lysate was isolated. The co-immunoprecipitation and Western blotting were carried out as previously described . Antibodies that were employed included monoclonal antibodies HA.11 (Covance), FLAG (M2, Sigma), SRF (Abcam) and an anti-acetyl-Lysine antibody (clone 4G12, Upsate); polyclonal antibodies HA (Santa Cruz), Flag (Sigma), a SRF antibody which recognizes the C-terminus of SRF protein (Santa Cruz) and p49/Strap .
The expression plasmids "EGFP-p49/STRAP" and "mCherry-NDUFAB1" were used for the transfection. At approximately 24, 48, and 72 hours after transfection, the expression of EGFP-p49/STRAP and mCherry-NDUFAB1 were examined by fluorescence microscopy using a Nikon Eclipse E600 equipped with CoolSNAP-ES digital camera and MetaVue 6.2 software.
The intensity of fluorescence was measured using the "ImageJ 1.37" program (National Institutes of Health, Bethesda, MD). Background noise was subtracted. The area of, and total fluorescence signal from, nuclear and non-nuclear cellular regions were measured separately. The cytoplasm superior and inferior to the nucleus was estimated to have a p49/STRAP concentration similar to that in the perinuclear region. It was further estimated that the cytoplasmic overlay on the nucleus was thinner than that of the surrounding region. For these reasons, the total fluorescence in the nuclear region was estimated to consist of p49 in the nucleus proper, and an amount of p49 represented by the product of the intensity of signal in the extranuclear region and the area of the nucleus itself.
Nuclear p49 signal = Total Nuclear Signal - (Extranuclear Signal Intensity × Nuclear Area)
The H9C2 cells were used for the assessment of NAD/NADH ratio in response to p49/STRAP gene expression. The NAD assay was performed using a protocol modified from the methods described by Zerez et al.  as well as Bernofsky and Swan , in which H9C2 cells were infected with 25 MOI of recombinant adenoviruses 48 hours before the assay was performed. The values are expressed as means ± S.D. A p value of less than 0.05 was considered to be significant.
NDUFAB1 is a p49/STRAP binding protein
p49/STRAP was first identified as a transcription cofactor of SRF . To explore a broad spectrum of potential p49/STRAP binding proteins, we employed a yeast two-hybrid system in which two overlapping cDNA fragments covering the entire p49 protein were used as baits to screen two human cDNA libraries. The utilization of separate bait fragments, p49/STRAP N-terminus (p49-N) and p49/STRAP C-terminus (p49-C), was intended to maximize the screening efficiency and to distinguish the binding proteins that may selectively interact with the different parts of the p49 protein.
A human heart cDNA library derived from terminally differentiated cardiomyocytes and non-cardiomyocyte cells, and a Hela cell cDNA library generated from highly proliferating tumor cells were selected to represent the cells with different biological features.
The NDUFAB1 protein selectively binds to the N-terminus of p49 protein (p49-N).
Origin of cDNA Library
Clones positive for NDUFAB1
To confirm the interaction between p49/STRAP and NDUFAB1 in the mammalian cells, we performed immunoprecipitation assays. As shown in figure 1b, immunoprecipitation of the endogenous p49/STRAP protein with anti-p49 antibody co-precipitated the NDUFAB1 protein. The expression of NDUFAB1 plasmid construct did not change the level of p49/STRAP protein in the cells (figure 1c).
Bioinformatics analysis of p49/STRAPand NDUFAB1
To determine the functional and conserved domains in p49/STRAP and NDUFAB1, we used web-based bioinformatic tools to analyze the protein sequences of these two genes.
Bioinformatics estimate of intracellular localization probability of p49 and NDUFAB1.
Vesicles of secretory
Analysis of NDUFAB1 revealed that NDUFAB1 was estimated to be 78.3% mitochondrial, 8.7% nuclear, 8.7% cytoplasmic and 4.3% cytoskeletal (table 2).
Intracellular localization of NDUFAB1
Intracellular localization of p49/STRAP
p49/STRAP expression affected NAD/NADH ratio
The p49/STRAP protein was initially isolated by our group as a transcription cofactor of SRF, which modulates the transcriptional regulation of SRF-target genes . In the present study, we found that p49/STRAP contained four classic NLS motifs. The p49 protein predominantly localized within the nucleus, but was also present at low levels in the cytoplasm. In addition, we found that p49/STRAP interacted with NDUFAB1, a subunit of the mitochondrial Complex I which regulates the redox status of NAD/NADH, and showed that NDUFAB1 could be observed in both the cytoplasm and nucleus. Furthermore, we found that overexpression of p49/STRAP altered the intracellular level of NAD and NADH, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also resulted in reduction of the acetylation of SRF. These results suggest that via interacting with a component of Complex I, p49/STRAP plays a role in the regulation of NAD and NADH which influence fundamental cellular processes such as cellular metabolism, gene expression, ion channel regulation and possibly aging [17, 18, 19].
It is known that Complex I, an enzyme complex that is located on the inner mitochondrial membrane catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ). In the redox process, the complex translocates electrons across the inner membrane, helping to build the electrochemical potential used to produce ATP . However, the exact catalytic mechanism remains incompletely established. Complex I is the largest respiratory complex, which contains 46 separate subunits. Of the 46 subunits, seven are encoded by the mitochondrial genome, while the rest are encoded by the nuclear genome.
NDUFAB1 is one of the nuclear-encoded subunits of Complex I, which contains a phosphopantetheine attachment site (DLGLDSLDQVEIIMAM), Characteristic of acyl carrier proteins. NDUFAB1 also contains an EF-hand calcium binding domain (DIDAEKLMCPQEI) . Deletion of NDUFAB1 homolog in N. crassa results in impaired assembly of Complex I and a four-fold increase in lysophospholipid content of mitochondrial membranes, suggesting that NDUFAB1 is required for the assembly of Complex I, and that it is also involved in fatty acid synthesis . Although NDUFAB1 was considered to be a membrane bound component of Complex I, recent evidence indicates that soluble NDUFAB1 protein has been found in the mitochondria, suggesting that NDUFAB1 may have other functions .
In the present study, NDUFAB1 was identified as a major binding protein to the p49/STRAP-N region. NDUFAB1 accounted for 95% of the cDNA clones derived from human heart and 40% of the cDNA clones derived from human Hela cells, which interacted with the p49-N region. NDUFAB1 mRNA is expressed in a wide range of human tissues. The highest expression levels are observed in adult heart, skeletal muscle and fetal heart . Although majority of the cDNA clones derived from human heart and Hela cells interacted with the p49-N region, none of the clones interacted with the p49-C region. These data suggest that the high binding rate of NDUFAB1 to p49/STRAP N-terminus is unlikely to be random, and is unlikely to be associated merely with the expression level of NDUFAB1 in the tissue origin of the cDNA library. Rather, this suggests that the NDUFAB1 protein binds selectively to the N-terminus, but not the C-terminus, of the p49/STRAP protein.
The translocation of a protein from the cytoplasm through the nuclear pore complex and into the nucleoplasm is usually initiated through the interaction between an NLS motif on the cargo protein with the NLS motif binding site on importin-α . The classic nuclear localization sequence generally conform to one of three types, known as pattern-4 (pat4), pattern-7 (pat7) (both are monopartite) and bipartite motifs [25, 26, 27]. The "pat4" NLS motif consists of a continuous stretch of four basic amino acids (lysine or arginine) or three basic amino acids associated with histidine or proline. The "pat7" NLS motif starts with a proline and is followed within three residues by an amino acid sequence containing three basic residues out of four. The third type of NLS motif, known as "bipartite" motif, consists of two basic amino acids, a 10 amino acid spacer and a five amino acid sequence containing at least three basic residues [25, 26, 27]. All four NLS motifs of the p49/STRAP protein comply with the classic definitions of NLS motifs. Our bioinformatic analyses also suggest that p49/STRAP could potentially localize in the other cellular compartments; likewise, NDUFAB1 may also be in the nucleus.
It is well documented that a single gene can give rise to more than one protein products with different sizes and with the potential to be localized to different intercellular compartments. These proteins can be derived from alternative splicing, or differential usage of translational initiation signals, thereby containing or lacking specific compartment targeting signals.
Recent studies indicate that even a single protein product can have the potential for dual intracellular localization. For example, wild-type p53 has been observed in cytoplasm and nucleus in both normal and tumor cells . p53 protein is accumulated in mitochondria and nuclei of cardiac myocytes after treatment with Adriamycin . Chronological observation of p53 protein revealed that upon TPA treatment, p53 was first translocated into mitochondria, and then into nucleus . Other examples of protein with dual localization include BCL2, SRF, ERK, Myopodin, 14-3-3 proteins, MKP-1 protein, thioredoxin and BRCA1 [31, 32, 33, 34, 35, 36, 37, 38].
It is considered that the localization signal sequence(s) in proteins play a major role in compartment localization of the proteins. Proteins that harbor two separate signals or an overlapping ambiguous signal, and even only one signal sequence may follow dual distribution in the cell . The mechanism of dual targeting is driven by the competition of various molecular events. Protein folding, post-translational modification and protein-protein interaction are key players in this phenomenon . Since proteins are generally considered to be translated in the cytosol, cytosol may be considered the default compartment for soluble proteins that do not transit to their initial programmed destination. The p49-N region contains three NLS motifs. Our data revealed that NDUFAB1 selectively interacted with p49-N region. It is possible that interaction of p49 with NDUFAB1 and other p49-binding protein(s) could make the NLS motifs of p49 protein unaccessible, thereby leading to the retention of p49 in the cytoplasm, as evidenced by the presence of p49 protein at low levels in the cytoplasm. Likewise, similar mechanisms could lead to the retention of NDUFAB1 in cytoplasm. It is also possible that the interaction between p49 and NDUFAB1 could retain both proteins within the cytoplasm, thus affecting their arrival at their programmed destination.
A recent study by Lisinski et al. reported that p49/STRAP is a cytosolic protein and was mainly localized to the cytoplasm . Apparently Lisinski and colleagues performed microscopic analysis on the cells three days after transfection assay. In the current study, the cells were analyzed at 24 hours, 48 hours and 72 hours after the transfection was initiated. p49/STRAP protein was observed in the nucleus in all of cells in which transfection was successful. However, p49 was present in both nucleus and cytoplasm in a minority of the cells. In addition, the fraction of total p49 protein accounted for by the cytosolic portion seemed to decrease, both in absolute value and variability, with the degree of confluency. Therefore, the differences between the two groups could be partly due to different culture conditions. It is also possible that the p49/STRAP protein could have differing localization under different conditions. Protein modifications including phosphorylation may also influence nuclear transport of p49/STRAP.
Inasmuch as p49/STRAP interacted with NDUFAB1 and had an impact on the intracellular NAD/NADH ratio, it is possible that the interaction affects the assembly of Complex I. Recently, it was reported that the transcription factor TFII-I plays a role outside the nucleus as an inhibitor of the cell surface Ca2+ channel TRPC3 by competing for binding to phospholipase C-g (PLC-g) . Similarly, p49/STRAP may also play a role outside of the nucleus. It is also possible that binding of NDUFAB1 by p49/STRAP could alter the ability of NDUFAB1 to enter mitochondria and/or could affect the assembling of Complex I, thereby altering its function and possibly the NAD/NADH ratio.
In this study, it was found that overexpression of p49/STRAP reduced the level of acetylated-SRF protein, suggesting that p49/STRAP may play a role in the regulation of SRF through the process of acetylation and deacetylation. Recent studies reveal that reversible acetylation of non-histone proteins, including transcription factors, play an important role in the regulation of gene expression. The reversible acetylation of non-histone proteins is usually carried out by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HATs and HDACs are often found to be binding proteins of a number of transcription factors and cofactors, and have also been found to be in protein complexes containing certain transcription factors and cofactors [42, 43]. In addition, certain transcription cofactors have been proven to be new members of a growing family of acetyltransferases and deacetylases)[42, 43, 44, 45]. It will be interesting to know the mechanisms by which p49/STRAP induces the deacetylation of the SRF protein. It is possible that p49/STRAP may function as a deacetylase that deacetylates SRF directly. It is also possible that p49/TRAP change the intracellular NAD/NADH ratio thereby activating (or recruiting) certain deacetylase(s) which in turn reduce the level of acetylated-SRF protein.
The findings in the present study demonstrate that p49/STRAP regulates intracellular NADH/NAD ratio and SRF deacetylation. The mechanism of regulation of NADH/NAD ratio by p49/STRAP is incompletely established at this time and remains to be further investigated. We plan to study the mechanism(s) in the future.
We are grateful to Dr. R. Tsien for m-Cherry red fluorescent protein, Dr. Vogelstein for AdEasy system. Supported in part by NIH grant #AG026091, The Central Arkansas Veterans Healthcare system, and the University of Arkansas for Medical Sciences.
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