Center for Biotechnology and Genomic Medicine
Center for Biotechnology and Genomic Medicine
Rao X.,Huazhong University of Science and Technology |
Rao X.,Center for Biotechnology and Genomic Medicine |
Zhong J.,Center for Biotechnology and Genomic Medicine |
Zhang S.,Huazhong University of Science and Technology |
And 7 more authors.
Circulation | Year: 2011
Background-: Despite intensive investigation, how DNA methylation influences endothelial function remains poorly understood. We used methyl-CpG-binding domain protein 2 (MBD2), an interpreter for DNA methylome-encoded information, to dissect the impact of DNA methylation on endothelial function in both physiological and pathophysiological states. Methods and results-: Human umbilical vein endothelial cells under normal conditions express moderate levels of MBD2, but knockdown of MBD2 by siRNA significantly enhanced angiogenesis and provided protection against H2O2-induced apoptosis. Remarkably, Mbd2 mice were protected against hind-limb ischemia evidenced by the significant improvement in perfusion recovery, along with increased capillary and arteriole formation. Loss of MBD2 activated endothelial survival and proangiogenic signals downstream of vascular endothelial growth factor signaling characterized by an increase in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor 2 expression, along with enhanced extracellular signal-regulated kinase 1/2 activation and BCL-2 expression. Mechanistic studies confirmed the methylation of CpG elements in the eNOS and vascular endothelial growth factor receptor 2 promoter. MBD2 binds to these methylated CpG elements and suppresses eNOS promoter activity. On ischemic insult, key endothelial genes such as eNOS and vascular endothelial growth factor receptor 2 undergo a DNA methylation turnover, and MBD2 interprets the changes of DNA methylation to suppress their expressions. Moreover, MBD2 modulation of eNOS expression is likely confined to endothelial cells because nonendothelial cells such as splenocytes fail to express eNOS after loss of MBD2. Conclusions-: We provided direct evidence supporting that DNA methylation regulates endothelial function, which forms the molecular basis for understanding how environmental insults (epigenetic factor) affect the genome to modify disease susceptibility. Because MBD2 itself does not affect the methylation of DNA and is dispensable for normal physiology in mice, it could be a viable epigenetic target for modulating endothelial function in disease states. © 2011 American Heart Association, Inc.
Pandey D.,Vascular Biology Center |
Chen F.,Vascular Biology Center |
Patel A.,Vascular Biology Center |
Wang C.-Y.,Center for Biotechnology and Genomic Medicine |
And 7 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2011
Objective-: Increased protein SUMOylation (small ubiquitin-related modifier [SUMO]) provides protection from cellular stress, including oxidative stress, but the mechanisms involved are incompletely understood. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress, and thus our goal was to determine whether SUMO regulates NADPH oxidase activity. Methods and Results-: Increased expression of SUMO1 potently inhibited the activity of Nox1 to Nox5. In contrast, inhibition of endogenous SUMOylation with small interfering RNA to SUMO1 or ubiquitin conjugating enzyme 9 or with the inhibitor anacardic acid increased ROS production from human embryonic kidney-Nox5 cells, human vascular smooth muscle cells, and neutrophils. The suppression of ROS production was unique to SUMO1, and it required a C-terminal diglycine and the SUMO-specific conjugating enzyme ubiquitin conjugating enzyme 9. SUMO1 did not modify intracellular calcium or Nox5 phosphorylation but reduced ROS output in an isolated enzyme assay, suggesting direct effects of SUMOylation on enzyme activity. However, we could not detect the presence of SUMO1 conjugation on Nox5 using a variety of approaches. Moreover, the mutation of more than 17 predicted and conserved lysine residues on Nox5 did not alter the inhibitory actions of SUMO1. Conclusion-: Together, these results suggest that SUMO is an important regulatory mechanism that indirectly represses the production of ROS to ameliorate cellular stress. © 2011 American Heart Association, Inc.
Sharma A.,Center for Biotechnology and Genomic Medicine |
Zhao J.,Center for Biotechnology and Genomic Medicine |
Podolsky R.,Center for Biotechnology and Genomic Medicine |
Podolsky R.,Georgia Regents University |
And 2 more authors.
Bioinformatics | Year: 2010
Motivation: Significance analysis of microarrays (SAM) is a widely used permutation-based approach to identifying differentially expressed genes in microarray datasets. While SAM is freely available as an Excel plug-in and as an R-package, analyses are often limited for large datasets due to very high memory requirements. Summary: We have developed a parallelized version of the SAM algorithm called ParaSAM to overcome the memory limitations. This high performance multithreaded application provides the scientific community with an easy and manageable client-server Windows application with graphical user interface and does not require programming experience to run. The parallel nature of the application comes from the use of web services to perform the permutations. Our results indicate that ParaSAM is not only faster than the serial version, but also can analyze extremely large datasets that cannot be performed using existing implementations. © The Author(s) 2010. Published by Oxford University Press.
Fang B.,H. Lee Moffitt Cancer Center and Research Institute |
Hoffman M.A.,H. Lee Moffitt Cancer Center and Research Institute |
Mirza A.-S.,H. Lee Moffitt Cancer Center and Research Institute |
Mirza A.-S.,University of South Florida |
And 11 more authors.
Methods | Year: 2015
Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. © 2015 Elsevier Inc.