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Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

In neurodegenerative diseases, extracellular vesicles (EVs) transfer pathogenic molecules and are consequently involved in disease progression. We have investigated the proteomic profiles of EVs that were isolated from four different human-induced pluripotent stem cell-derived neural cell types (excitatory neurons, astrocytes, microglia-like cells, and oligodendrocyte-like cells). Novel cell type-specific EV protein markers were then identified for the excitatory neurons (ATP1A3, NCAM1), astrocytes (LRP1, ITGA6), microglia-like cells (ITGAM, LCP1), and oligodendrocyte-like cells (LAMP2, FTH1), as well as 16 pan-EV marker candidates, including integrins and annexins. To further demonstrate how cell-type-specific EVs may be involved in Alzheimer's disease (AD), we performed protein co-expression network analysis and conducted cell type assessments for the proteomes of brain-derived EVs from the control, mild cognitive impairment, and AD cases. A protein module enriched in astrocyte-specific EV markers was most significantly associated with the AD pathology and cognitive impairment, suggesting an important role in AD progression. The hub protein from this module, integrin-β1 (ITGB1), was found to be significantly elevated in astrocyte-specific EVs enriched from the total brain-derived AD EVs and associated with the brain β-amyloid and tau load in independent cohorts. Thus, our study provides a featured framework and rich resource for the future analyses of EV functions in neurodegenerative diseases in a cell type-specific manner.

Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle4-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes.sup.1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat.sup.4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle. Tissue nonspecific alkaline phosphatase (TNAP) within mitochondria hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation in thermogenic fat cells., Author(s): Yizhi Sun [sup.1] [sup.2] , Janane F. Rahbani [sup.3] [sup.4] , Mark P. Jedrychowski [sup.1] [sup.2] , Christopher L. Riley [sup.1] [sup.2] , Sara Vidoni [sup.1] [sup.2] , Dina [...]

Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

The sympathetic nervous system innervates peripheral organs to regulate their function and maintain homeostasis, whereas target cells also produce neurotrophic factors to promote sympathetic innervation.sup.1,2. The molecular basis of this bi-directional communication remains to be fully determined. Here we use thermogenic adipose tissue from mice as a model system to show that T cells, specifically [gamma][delta] T cells, have a crucial role in promoting sympathetic innervation, at least in part by driving the expression of TGF[beta]1 in parenchymal cells via the IL-17 receptor C (IL-17RC). Ablation of IL-17RC specifically in adipose tissue reduces expression of TGF[beta]1 in adipocytes, impairs local sympathetic innervation and causes obesity and other metabolic phenotypes that are consistent with defective thermogenesis; innervation can be fully rescued by restoring TGF[beta]1 expression. Ablating [gamma][delta] [TAU] cells and the IL-17RC signalling pathway also impairs sympathetic innervation in other tissues such as salivary glands. These findings demonstrate coordination between T cells and parenchymal cells to regulate sympathetic innervation. V[gamma]6.sup.+ V[delta]1.sup.+ [gamma][delta] T cells control tolerance to cold by activating adipocyte IL-17RC and promoting sympathetic innervation of thermogenic adipose tissue in mice., Author(s): Bo Hu [sup.1] [sup.2] , Chengcheng Jin [sup.3] [sup.6] , Xing Zeng [sup.1] [sup.2] , Jon M. Resch [sup.4] , Mark P. Jedrychowski [sup.1] [sup.2] , Zongfang Yang [sup.4] [...]

Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phosphoregulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson's disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and noncanonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN's ability to assemble canonical and noncanonical UB chains and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feedforward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage.

Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease.sup.1. Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP-protein kinase A signalling; this pathway has been subject to longstanding clinical investigation.sup.2-4. Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule.A comparative metabolomics approach is used to identify succinate as a key activator of thermogenesis in brown adipose tissue., Author(s): Evanna L. Mills [sup.1] [sup.2] , Kerry A. Pierce [sup.3] , Mark P. Jedrychowski [sup.1] [sup.2] , Ryan Garrity [sup.1] , Sally Winther [sup.1] [sup.2] , Sara Vidoni [sup.1] [...]

Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes), scaffolded by hematopoietic protein 1 (Hem-1), that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE)2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a) dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b) substantially weakens Rac activation and phosphatidylinositol-(3,4,5)-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5)-tris-phosphate)/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c) prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1) (1,2). Thermogenesis from these adipocytes can combat obesity and diabetes (3), encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders., The specialized capacity for thermogenic respiration in brown adipose tissue (BAT) and beige fat (4) relies on UCP1. This mitochondrial inner membrane carrier dissipates protonmotive force (Δp) and increases the [...]