OBJECTIVE-To examine fat biopsy samples from lean insulinsensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress.RESEARCH DESIGN AND METHODS-Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel and MALDI-TOF/TOF), Western blot, and RT-PCR analysis.RESULTS-Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress-related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P. Western blotting revealed upregulation of several other UPR stress-related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH 2 -terminal kinase (JNK)-1, a downstream effector protein of ER stress. RT-PCR analysis revealed upregulation of the spliced form of X-box binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway.CONCLUSIONS-These findings represent the first demonstration of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation. Diabetes 57: [2438][2439][2440][2441][2442][2443][2444] 2008 O besity is associated with insulin resistance and with a low-grade state of inflammation (1). Whereas the cause of neither is completely understood, there is good evidence to show that free fatty acids (FFAs) play an important role in the development of obesity-related insulin resistance and inflammation (2). Plasma FFA levels are increased in most obese people (3). Acutely raising plasma FFA levels increases insulin resistance (4), whereas lowering plasma FFA levels reduces insulin resistance (5). Mechanisms involved in FFAinduced insulin resistance include accumulation (in muscle and liver) of lipids and lipid intermediates, including diacylglycerol; activation of several protein kinase C isoforms; and reduction in tyrosine phosphorylation of insulin receptor substrate-1/2 (6 -8). FFAs also activate the proinflammatory nuclear factor B pathway (6,9), in part, via signaling through toll-like receptor-4 pathways (10). However, not all obese, insulin-resistant subjects have elevated plasma FFA levels. It is therefore likely that there are other causes for obesityrelated insulin resistance. One of these appears to be endoplasmic reticulum (ER) stress. Indeed, chronic excessive nutrient intake has been shown to cause ER stress in adipose tissue of ob/ob mice and mice fed high-fat diets (11-13).The ER is a major site for protein as well as for lipid and sterol synthesis (14,15). Ribosomes attached to the ER membranes release newly synthesized peptides into the ER lumen, where protein chaperones and foldases assist in the proper posttranslational modification a...

SUMMARY: Cyclin-Dependent Kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50=5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

SUMMARY Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1, MCUR1, have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation and migration but elicited autophagy. These studies establish the existence of a MCU complex which assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.

Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (< 24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD.

Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2␣ expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2␣. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics.