Correlation of Levels of Folded Recombinant p53 in Escherichia coli with Thermodynamic Stability in Vitro

The amount of folded functional protein in a cell is controlled by a number of factors, including the relative rates of its biosynthetic and specific degradation processes, and its intrinsic thermodynamic stability. Mutationinduced loss of stability is a common cause of disease. Many oncogenic mutants of the tumour suppressor p53, for example, reduce the intrinsic thermodynamic stability of the protein in vitro. We have analysed the level of recombinant folded human p53 core domain (p53C) and its mutants in Escherichia coli spanning a stability range of 6 kcal/mol to assess the effects of intrinsic thermodynamic stability in vivo in the absence of specific ubiquitin-mediated pathways in human cells. The levels of folded protein were measured fluorimetrically in living cells by fusing the gene of p53C upstream to that of green fluorescent protein and measuring the fluorescence relative to a control at various temperatures. At a fixed temperature, the amount of fluorescence is correlated with the thermodynamic stability of the mutant. The level of each protein varied with temperature according to a sigmoid curve that paralleled the melting in vitro, but the apparent Tm was lower in vivo, because steady-state levels are observed rather than true thermodynamic equilibria. Our results show clearly that changes in the intrinsic thermodynamic stability of p53 reduce the level of folded and hence functional p53 substantially in E. coli, and provide insights into the correlation between protein instability and disease at the cellular level

Pan SMARCA PB1 Bromodomain Inhibitors and Their Role in Regulating Adipogenesis

Accessibility of the human genome is modulated by the ATP driven SWI SNF chromatin remodeling multiprotein complexes BAF BRG1 BRM associated factor and PBAF polybromo associated BAF factor , which involves reading of acetylated histone tails by the bromodomain containing proteins SMARCA2 BRM , SMARCA4 BRG1 , and polybromo 1. Dysregulation of chromatin remodeling leads to aberrant cell proliferation and differentiation. Here, we have characterized a set of potent and cell active bromodomain inhibitors with pan selectivity for canonical family VIII bromodomains. Targeted SWI SNF bromodomain inhibition blocked the expression of key genes during adipogenesis, including the transcription factors PPAR amp; 947; and C EBP amp; 945;, and impaired the differentiation of 3T3 L1 murine fibroblasts into adipocytes. Our data highlight the role of SWI SNF bromodomains in adipogenesis and provide a framework for the development of SWI SNF bromodomain inhibitors for indirect targeting of key transcription factors regulating cell differentiatio

Development of a Selective Dual Discoidin Domain Receptor DDR p38 Kinase Chemical Probe

Discoidin domain receptors 1 and 2 DDR1 2 play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so called chemical probe compounds, have been developed to study DDR1 2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen activated protein kinase inhibitor 7 toward a potent and cell active dual DDR p38 chemical probe and developed a structurally related negative control. The structure guided design approach used provided insights into the P loop folding process of p38 and how targeting of non conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR p38 probe, 30 SR 302 , is a valuable tool for studying the role of DDR kinase in normal physiology and in disease developmen