Converting CooA from a Carbon Monoxide to an Oxygen-Sensing Heme Protein Transcription Factor: Investigations into the Structure and Mechanism of Gas Binding

CooA is a carbon monoxide-sensing (CO-sensing) heme protein transcription factor that regulates gene activation in several bacteria and, importantly, is a convenient model for studying analogous proteins in the human body. In the present study, the specificity and mechanism of gas-binding of CooA have been investigated by efforts to convert CooA from a CO to an oxygen (O2) sensor through site directed mutagenesis of residues in the gas binding pocket of the heme group. The resulting mutated proteins were then isolated and characterized with spectroscopy. The results of this research project will provide further insight into the current model for the specificity and mechanisms of gas binding in heme proteins

Hypoxia induces differential translation of enolase/MBP-1

<p>Abstract</p> <p>Background</p> <p>Hypoxic microenvironments in tumors contribute to transformation, which may alter metabolism, growth, and therapeutic responsiveness. The α-enolase gene encodes both a glycolytic enzyme (α-enolase) and a DNA-binding tumor suppressor protein, c-myc binding protein (MBP-1). These divergent α-enolase gene products play central roles in glucose metabolism and growth regulation and their differential regulation may be critical for tumor adaptation to hypoxia. We have previously shown that MBP-1 and its binding to the c-myc P₂promoter regulates the metabolic and cellular growth changes that occur in response to altered exogenous glucose concentrations.</p> <p>Results</p> <p>To examine the regulation of α-enolase and MBP-1 by a hypoxic microenvironment in breast cancer, MCF-7 cells were grown in low, physiologic, or high glucose under 1% oxygen. Our results demonstrate that adaptation to hypoxia involves attenuation of MBP-1 translation and loss of MBP-1-mediated regulation of c-myc transcription, evidenced by decreased MBP-1 binding to the c-myc P₂promoter. This allows for a robust increase in c-myc expression, "early c-myc response", which stimulates aerobic glycolysis resulting in tumor acclimation to oxidative stress. Increased α-enolase mRNA and preferential translation/post-translational modification may also allow for acclimatization to low oxygen, particularly under low glucose concentrations.</p> <p>Conclusions</p> <p>These results demonstrate that malignant cells adapt to hypoxia by modulating α-enolase/MBP-1 levels and suggest a mechanism for tumor cell induction of the hyperglycolytic state. This important "feedback" mechanism may help transformed cells to escape the apoptotic cascade, allowing for survival during limited glucose and oxygen availability.</p

MALDI-MSI and label-free LC-ESI-MS/MS shotgun proteomics to investigate protein induction in a murine fibrosarcoma model following treatment with a vascular disrupting agent

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents (VDAs) and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response bio-markers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate (CA4P) was examined in a mouse fibrosarcoma model (fs 188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by CA4P and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-MSI. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical (IHC) staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners

c-Myc quadruplex-forming sequence Pu-27 induces extensive damage in both telomeric and nontelomeric regions of DNA

Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66-76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G2 checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H2AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H2AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex

Effect of sex and RYR1 gene mutation on the muscle proteomic profile and main physiological biomarkers in pigs at slaughter

Gender and RYR1 gene mutation might have an effect on the muscle metabolic characteristics and on the animal's stress at slaughter, which could influence the process of muscle-to-meat conversion. Forty-eight pigs were distributed in a design including two factors: sex (male/female) and RYR1 genotype (NN/Nn). At slaughter, physiological blood biomarkers and muscle proteome were analyzed and carcass and meat quality traits were registered. Females had higher serum levels of glucose, urea, C-reactive protein "CRP", Pig-MAP and glutation-peroxidase "GPx" and lower levels of lactate, showed faster muscle pH decline and higher meat exudation. RYR1 mutation increased serum creatinine, creatine kinase and CRP and decreased GPx. The proteomic study highlighted significant effects of gender and RYR1 genotype on proteins related to fibre composition, antioxidant defense and post mortem glycolytic pathway, which correlate to differences of meat quality. This study provides interesting information on muscle biomarkers of the ultimate meat quality that are modulated by the animal's individual susceptibility to stress at slaughter.info:eu-repo/semantics/acceptedVersio

Abstract 5193: The importance of primo vascular system (PVS) on cancer metastasis and therapeutic intervention: A new dimension of cancer stem cell

Abstract The ability of cancer cells especially immature and multi-potent cancer stem cells to escape from conventional therapeutic intervention has been long investigated. The exact mechanisms and safe harboring of this population of cells have yet to be identified. The primo vascular system (PVS) which is composed of new types of micro-conduits named primo-vessels (PV) and primo-nodes (PN) has recently emerged as a third component of circulatory system. In this study, we have shown the existence of PVS and their networks in murine xenograft of human histiocytic lymphoma cells (U937) using trypan blue. These PVs are thread-like, elastic, multi-lumen structure and appears to radiate from PN, perpendicular of the conventional venuoles-arteriols and reside in close proximity to the tumor. PNs are about 500-600 μM membranous sac-like structures, containing numerous tiny cells evident by DAPI staining. Isolation of RNA from PVS signifies the presence of intact transcriptional machinery in the cells. Hematoxylin and Eosin (H&amp;E) staining of PVS shows the presence of loose and abortive structure lined by fibroblast but filled with lightly dense fibers, cells, lacuna and nerve-like structures. To further characterize the origin and type of cells within the PVS, immunostaining with antibodies for CD68, CD45 and lysozyme has revealed the cells are same immunophenotype as of U937. RT-PCR arrays demonstrates a hundred to thousand fold up regulation of human cancer stem cell specific transcription factors most notably KLF4, an upstream regulator of NANOG which maintain the pluripotent and undifferentiated state of stem cells. These results suggest that PVS close to the tumor could be the safe haven for a selective population of cancer stem cell. Further understanding of the biological properties and responsiveness to therapy of these cells will shed light on new dimensions of anti-cancer interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5193. doi:1538-7445.AM2012-5193</jats:p