Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.
Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecules of a protein crystal but absent in others, or present in some structures of a protein but absent in others. We reasoned that the labile nature of these disulfides signifies a propensity for cleavage and so possible allosteric regulation of the protein in which the bond resides. A total of 511 labile disulfide bonds were identified. The labile disulfides are more stressed than the average bond, being characterized by high average torsional strain and stretching of the sulfur–sulfur bond and neighbouring bond angles. This pre-stress likely underpins their susceptibility to cleavage. The coagulation, complement and oxygen-sensing hypoxia inducible factor-1 pathways, which are known or have been suggested to be regulated by allosteric disulfides, are enriched in proteins containing labile disulfides. The identification of labile disulfide bonds will facilitate the study of this post-translational modification.
The impact of aerobic training on human skeletal muscle cell (HSkMC) mitochondrial metabolism is a significant research gap, critical to understanding the mechanisms by which exercise augments skeletal muscle metabolism. We therefore assessed mitochondrial content and capacity in fully differentiated CD56+ HSkMCs from lean active (LA) and sedentary individuals with obesity (OS) at baseline, as well as lean/overweight sedentary individuals (LOS) at baseline and following an 18-day aerobic training intervention. Participants had in vivo skeletal muscle PCr recovery rate by 31P-MRS (mitochondrial oxidative kinetics) and cardiorespiratory fitness (VO2max) assessed at baseline. Biopsies of the vastus lateralis were performed for the isolation of skeletal muscle stem cells. LOS individuals repeated all assessments post-training. HSkMCs were evaluated for mitochondrial respiratory capacity by high resolution respirometry. Data were normalized to two indices of mitochondrial content (CS activity and OXPHOS protein expression) and a marker of total cell count (quantity of DNA).LA individuals had significantly higher VO2max than OS and LOS-Pre training; however, no differences were observed in skeletal muscle mitochondrial capacity, nor in carbohydrate- or fatty acid-supported HSkMC respiratory capacity. Aerobic training robustly increased in vivo skeletal muscle mitochondrial capacity of LOS individuals, as well as carbohydrate-supported HSkMC respiratory capacity. Indices of mitochondrial content and total cell count were similar among the groups and did not change with aerobic training.Our findings demonstrate that bioenergetic changes induced with aerobic training in skeletal muscle in vivo are retained in HSkMCs in vitro without impacting mitochondrial content, suggesting that training improves intrinsic skeletal muscle mitochondrial capacity.
Our group previously demonstrated that sarcoma cell lines were insensitive to epidermal growth factor receptor (EGFR) inhibitor gefitinib monotherapy. PENAO, an anti-tumour metabolic compound created in our laboratory, is currently in clinical trials. Considering the positive regulation of tumour energy production by both the EGFR signalling and tumour metabolism pathways, this study aimed to investigate the effect and mechanisms of combination therapy using gefitinib and PENAO in sarcoma cell lines in vitro and in vivo. PENAO monotherapy reduced proliferation in 12 sarcoma cell lines. Combining gefitinib and PENAO resulted in synergistic inhibition in both a time-and dosedependent manner in 3 sarcoma cell lines with less prominent monotherapy effects. Combined treatment significantly enhanced cell death and perturbed mitochondrial function. In vivo combination therapy with PENAO and gefitinib was non-toxic to mice and significantly delayed tumour growth and prolonged survival. At 20 days after treatment, tumours from the combination treated mice were significantly smaller than those from untreated and single drug treated mice. The survival curves also showed significant difference across and between groups. The combination of PENAO and gefitinib in vitro and in vivo, shows promise as a treatment pathway in this poor outcome tumour.
AEs in kidney transplant immunosuppression trials appear to be selectively reported and may be unreliable for clinical decisions. Adherence to the Consolidated Standards of Reporting Trials harms extension should be mandatory to ensure transparent reporting of AEs that are important to patients and clinicians.
Aims/Hypothesis: Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous; however, this association remains controversial. The aim of this study was to perform an in-depth multi-factorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active), individuals with obesity (Obese) and individuals with Obesity, insulin resistance and type 2 diabetes (T2D). Methods: Skeletal muscle biopsies were obtained from the Vastus Lateralis of individuals who were lean and active (Active- n = 9), individuals with obesity (Obese- n = 9) and individuals with obesity insulin resistance and T2D (T2D- n =22) in this cross-sectional design. Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by BN-PAGE and immunoblot. TCA cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. Results: Active had greater mitochondrial capacity compared to both Obese and T2D for ex vivo mitochondrial respiration with fatty-acid and glycolytic supported protocols adjusted for mitochondrial content (P < 0.05). Complex IV supercomplex assembley was greater in Active compared to Obese and T2D (P < 0.05) whereas Complex I and III supercomplex assembly was greater in Active compared to T2D only (P < 0.05). TCA cycle intermediates; citrate, succinate, fumarate and malate were all significantly greater in Active compared to Obese and T2D (P < 0.05). Strikingly, Obese and T2D do not differ in any of the skeletal muscle mitochondrial measurements. Active had an upregulation of genes related to respiration/mitochondrial capacity compared to both Obese and T2D. Transcriptional differences between Obese and T2D were not driven by mitochondrial related process. Active had reduced methylation correlated with increased gene expression for important mitochondrial-related genes, including ATP5PD and MFN2. Conclusions/Interpretations: We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity) that affect mitochondrial capacity. We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance.
Inter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. Beginning with the discovery of insulin over a century ago, characterization of molecules responsible for signal between tissues has required careful and elegant experimentation where these observations have been integral to deciphering physiology and disease. Given that circulating bioactive factors are stable in serum, occur naturally, and are easily assayed from blood, they present obvious focal molecules for therapeutic intervention and biomarker development. For example, physiologic dissection of the actions of soluble proteins such as proprotein convertase subtilisin/kexin type 9 (PCSK9) and glucagon-like peptide 1 (GLP1) have yielded among the most promising therapeutics to treat cardiovascular disease and obesity, respectively 1–4 . A major obstacle in the characterization of such soluble factors is that defining their tissues and pathways of action requires extensive experimental testing in cells and animal models. Recently, studies have shown that secreted proteins mediating inter-tissue signaling could be identified by “brute-force” surveys of all genes within RNA-sequencing measures across tissues within a population 5–9 . Expanding on this intuition, we reasoned that parallel strategies could be leveraged to understand how individual genes mediate signaling across metabolic tissues through correlative analysis of genetic variation. Thus, genetics could aid in understanding cross-organ signaling by adopting a genecentric approach. Here, we surveyed gene-gene genetic correlation structure for ∼6.1×10^ 12 gene pairs across 18 metabolic tissues in 310 individuals where variation of genes such as FGF21, ADIPOQ, GCG and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both local signaling mechanisms (liver PCSK9) as well as genes encoding enzymes producing metabolites (adipose PNPLA2), where genetic correlation structure aligned with known roles for these critical metabolic pathways. Finally, we utilized this resource to suggest new functions for metabolic coordination between organs. For example, we prioritized key proteins for putative signaling between skeletal muscle and hippocampus, and further suggest colon as a central coordinator for systemic circadian clocks. We refer to this resource as G enetically- D erived C orrelations A cross Tissues (GD-CAT) where all tools and data are built into a web portal enabling users to perform these analyses without a single line of code (gdcat.org). This resource enables querying of any gene in any tissue to find genetic coregulation of genes, cell types, pathways and network architectures across metabolic organs.
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