The effect of different training modes on skeletal muscle microvascular density and endothelial enzymes controlling NO availability

It is becoming increasingly apparent that a high vasodilator response of the skeletal muscle microvasculature to insulin and exercise is of critical importance for adequate muscle perfusion and long-term microvascular and muscle metabolic health. Previous research has shown that a sedentary lifestyle, obesity, and ageing lead to impairments in the vasodilator response, while a physically active lifestyle keeps both microvascular density and vasodilator response high. To investigate the molecular mechanisms behind these impairments and the benefits of exercise training interventions, our laboratory has recently developed quantitative immunofluorescence microscopy methods to measure protein content of eNOS and NAD(P)Hoxidase specifically in the endothelial layer of capillaries and arterioles of human skeletal muscle. As eNOS produces NO and NAD(P)Hoxidase superoxide anions (quenching NO) we propose that the eNOS/NAD(P)Hoxidase protein ratio is a marker of vasodilator capacity. The novel methods show that endurance training (ET) and high intensity interval training (HIT) generally regarded as a time efficient alternative to ET, increase eNOS protein content and the eNOS/NADP(H) oxidase protein ratio in previously sedentary lean and obese young men. Resistance exercise training had smaller but qualitatively similar effects. Western blot data of other laboratories suggest that endurance exercise training leads to similar changes in sedentary elderly men. Future research will be required to investigate the relative importance of other sources and tissues in the balance between NO and O2- production seen by the vascular smooth muscle layer of terminal arterioles

Nitric oxide is required for the insulin sensitizing effects of contraction in mouse skeletal muscle

The factors regulating the increase in skeletal muscle insulin sensitivity after exercise are unclear. We examined whether nitric oxide (NO) is required for the increase in insulin sensitivity after ex vivo contractions. Isolated C57BL/6J mouse EDL muscles were contracted for 10min or remained at rest (basal) with or without the NO synthase (NOS) inhibition (N-G-monomethyl-l-arginine; l-NMMA; 100m). Then, 3.5h post contraction/basal, muscles were exposed to saline or insulin (120Uml(-1)) with or without l-NMMA during the last 30min. l-NMMA had no effect on basal skeletal muscle glucose uptake. The increase in muscle glucose uptake with insulin (57%) was significantly (P<0.05) greater after prior contraction (140% increase). NOS inhibition during the contractions had no effect on this insulin-sensitizing effect of contraction, whereas NOS inhibition during insulin prevented the increase in skeletal muscle insulin sensitivity post-contraction. Soluble guanylate cyclase inhibition, protein kinase G (PKG) inhibition or cyclic nucleotide phosphodiesterase inhibition each had no effect on the insulin-sensitizing effect of prior contraction. In conclusion, NO is required for increases in insulin sensitivity several hours after contraction of mouse skeletal muscle via a cGMP/PKG independent pathway

Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production

Pharmacologic Tumor PDL1 Depletion with Cefepime or Ceftazidime Promotes DNA Damage and Sensitivity to DNA-Damaging Agents

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the β-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The β-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy

Pharmacologic Tumor PDL1 Depletion with Cefepime or Ceftazidime Promotes DNA Damage and Sensitivity to DNA-Damaging Agents

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the β-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The β-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy.</jats:p

Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A’s deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A’s potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions