Project description:Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool in the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar to temperature gradients, increasing concentrations of organic solvents stimulate unfolding and precipitation of the cellular proteome. This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al., 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves. We readily identified the specific targets of compounds with known mechanisms of action. As a further efficiency boost, we applied the concept of area-under-the-curve analysis to develop solvent proteome integral solubility alteration (solvent-PISA) and demonstrate that this approach can serve as a reliable surrogate for SPP. We propose that by combining SPP with alternative methods, like thermal proteome profiling, it will be possible to increase the absolute number of high-quality melting curves that are attainable by either approach individually thereby increasing the fraction of the proteome that can be screened for evidence of ligand binding.
Project description:Physical activity is thought to provide clinical benefit in Parkinson’s diseas (PD). Irisin is a blood-brain barrier permeable exercise-induced polypeptide secreted by muscle that mediates, in part, the beneficial effects of exercise. Here we show that irisin prevents pathologic -synuclein (-syn) induced neurodegeneration in the -syn preformed fibril mouse model of sporadic PD. Intravenous delivery of adenoviral irisin in vivo after the stereotaxic intrastriatal injection of -syn pre-formed fibrils reduced the formation of pathologic -syn and prevented the loss of dopamine neurons and reductions in striatal dopamine. Irisin also reduced the -syn pre-formed fibril induced motor deficits as assessed by the pole test and grip strength test. Administration of recombinant irisin in primary cortical neurons prevented pathologic -syn toxicity. Tandem mass spectrometry and biochemical analysis revealed that irisin reduced pathologic -syn by enhancing endolysosomal degradation of pathologic -syn. Our findings highlight the potential for therapeutic disease modification of irisin in PD.
Project description:Obesity is associated with systemic inflammation that impairs mitochondrial function. These changes curtail oxidative metabolism, limiting adipocyte lipid metabolism and thermogenesis, a metabolically beneficial program that dissipates chemical energy as heat. Here, we show that PGC1⍺, a key governor of mitochondrial biogenesis, is negatively regulated at the level of its mRNA translation by the RNA-binding protein RBM43. RBM43 is induced by inflammatory cytokines and suppresses mitochondrial biogenesis in a PGC1⍺-dependent manner. In mice, adipocyte-selective Rbm43 disruption elevates PGC1⍺ translation and oxidative metabolism. In obesity, Rbm43 loss confers body weight-independent protection from glucose intolerance, adipose inflammation, and activation of the innate immune sensor cGAS-STING. We further identify a role for PGC1⍺ in safeguarding against cytoplasmic accumulation of mitochondrial DNA, a cGAS ligand. The action of RBM43 defines a translational regulatory pathway by which inflammatory signals dictate cellular energy metabolism and contribute to metabolic disease pathogenesis.
Project description:Obesity is associated with systemic inflammation that impairs mitochondrial function. These changes curtail oxidative metabolism, limiting adipocyte lipid metabolism and thermogenesis, a metabolically beneficial program that dissipates chemical energy as heat. Here, we show that PGC1⍺, a key governor of mitochondrial biogenesis, is negatively regulated at the level of its mRNA translation by the RNA-binding protein RBM43. RBM43 is induced by inflammatory cytokines and suppresses mitochondrial biogenesis in a PGC1⍺-dependent manner. In mice, adipocyte-selective Rbm43 disruption elevates PGC1⍺ translation and oxidative metabolism. In obesity, Rbm43 loss confers body weight-independent protection from glucose intolerance, adipose inflammation, and activation of the innate immune sensor cGAS-STING. We further identify a role for PGC1⍺ in safeguarding against cytoplasmic accumulation of mitochondrial DNA, a cGAS ligand. The action of RBM43 defines a translational regulatory pathway by which inflammatory signals dictate cellular energy metabolism and contribute to metabolic disease pathogenesis.
Project description:The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic breast cancer cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this dataset with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex dataset by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.
Project description:Proteins are secreted from various cells to send information to neighboring cells or distant tissues. Because of the highly integrated nature of energy balance systems, there has been a great deal of interest in myokines and adipokines, proteins secreted from muscle and fat tissues, respectively. These have been challenging to study through proteomics because serum is loaded with super-abundant proteins that limit the detection of proteins that have a low, hormone-like abundance. We show here that interstitial fluids (IFs) can be harvested easily from muscle and fat tissues of mice by very low-speed centrifugation and these fluids show a very different protein constellation than that of plasma or tissues. Under several different perturbations in vivo, like exercise or cold, quantitative Mass Spectrometry of these IFs allowed for the identification of many novel myokines and adipokines, including factors both increased and decreased in abundance. Finally, we identify prosaposin, a well-known neurotrophic factor, as a secreted product of both muscle and fat tissues; recombinant prosaposin stimulates a gene program related to thermogenesis in primary fat cells, indicating one potential function for PSAP.
Project description:Type 2 diabetes (T2D) is major cause of mortality and morbidity. The key manifestations of this disease are insulin resistance (IR), hyperglycemia and hyperinsulinemia. Secretion of insulin from the endocrine pancreas is triggered by elevated circulating glucose and acts on skeletal muscle and adipose tissues to remove excess glucose from the blood (1). This is achieved through a series of intracellular events that trigger the translocation of vesicles containing the glucose transporter 4 (GLUT4) to the surface of skeletal muscle cells and adipocytes (2,3). This process is impaired in patients with IR or T2D and insulin fails to promote GLUT4 translocation, thus resulting in inefficient glucose uptake in storage tissue and hyperglycemia (4). Chronic hyperglycemic condition increases the risk of cardiovascular disease, stroke, neuropathy, and death (5). Previously developed insulin sensitizer like thiazolidinediones (TZDs) have proven effective in improving glycemic control, however, they significantly increase the risk of cardiovascular disease due to their agonist activity at PPAR(6-8). Consequently, identifying insulin sensitizers that restore insulin-stimulated GLUT4 translocation in diabetic patients without targeting PPAR is urgently needed. The discovery of such molecules has been challenging due to the lack of a GLUT4 translocation assay amenable to high throughput screening (HTS). For this reason, we have developed a new luminescence based HTS assay that allows real time monitoring of GLUT4 translocation in mammalian cells and further adapted the assay to measure GLUT4 translocation in live mice. Using this assay, we identified a new insulin sensitizer which greatly improves insulin-dependent glucose uptake in storage tissues and glucose tolerance in insulin resistant rodents. This new insulin sensitizers acts through interaction with the Unc119 family of proteins which are known to facilitate the transport of specific cargos (9) but had not yet been implicated in insulin action or GLUT4 translocation. This study identifies new PPAR-independent insulin sensitizers with in-vivo efficacy and identify Unc119 proteins as new targets for the treatment of T2D.
Project description:We show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to the extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. Loss of Mustn1 in vascular smooth muscle cells does not affect their proliferative or migratory functions. We show that Mustn1 can be secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle interstitial fluid, in particular during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates.
Project description:The mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential mostly by inducing the expression of the electron transport chain but also activates the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited by either phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of the mitochondrial membrane potential is evolutionarily conserved as it also is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that the mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement even under conditions of mitochondrial dysfunction.
Project description:Mass spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground truth phosphorylation positions that can serve as gold standards for method development and pipeline evaluation. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides produced in E. coli using the phosphoserine orthogonal translation system. Because phosphoserine is genetically encoded, the position of phosphorylation is precisely known. Since the library is synthesized in E. coli, phosphopeptide production is scalable and regenerable. We demonstrate the utility of iSPI as a phosphopeptide standard to investigate instrument acquisition methods, to evaluate and optimize phosphorylation site localization algorithms, and to compare performance across data analysis pipelines. As a direct result of iSPI analyses, we present AscorePro, an updated version of the Ascore algorithm that has been specifically optimized for localization of phosphorylation sites in higher energy fragmentation spectra. In addition, we provide the FLR Viewer for Phosphorylation Site Localization, a browser-based tool allowing the community to calculate phosphorylation site false localization rates using their own workflows and analysis pipelines. Thus, iSPI and its associated data constitute a useful, multi-dimensional resource for the phosphoproteomics community.