GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

Systematic evaluation of AML-associated antigens identifies anti-U5 SNRNP200 therapeutic antibodies for the treatment of acute myeloid leukemia.

Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.

Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels.

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

Chronic UCN2 treatment desensitizes CRHR2 and improves insulin sensitivity.

Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport into muscle and adipocytes, central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter in the plasma membrane ( PM ). Physiologic signals (activated insulin receptor or AMP kinase [ AMPK ]), acutely increase PM GLUT4 to enhance glucose uptake. Here we show in kinetic studies that intracellular GLUT4 is in equilibrium with the PM in unstimulated cultured human skeletal muscle cells, and that AMPK promotes GLUT4 redistribution to the PM by regulating both exocytosis and endocytosis. AMPK-stimulation of exocytosis requires Rab10 and Rab GTPase activating protein TBC1D4, requirements shared with insulin control of GLUT4 in adipocytes. Using APEX2 proximity mapping, we identify, at high-density and high-resolution, the GLUT4 proximal proteome, revealing GLUT4 traverses both PM proximal and distal compartments in unstimulated muscle cells. These data support intracellular retention of GLUT4 in unstimulated muscle cells by a dynamic mechanism dependent on the rates of internalization and recycling. AMPK promoted GLUT4 translocation to the PM involves redistribution of GLUT4 among the same compartments traversed in unstimulated cells, with a significant redistribution of GLUT4 from the PM distal Trans Golgi Network Golgi compartments. The comprehensive proximal protein mapping provides an integrated, whole cell accounting of GLUT4's localization at a resolution of ∼20 nm, a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in physiologically relevant cell type and as such, sheds new light on novel key pathways and molecular components as potential therapeutic approaches to modulate muscle glucose uptake.

Proteomic Profiling of Intra-Islet Features Reveals Substructure-Specific Protein Signatures.

Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.

DUX4 expression activates JNK and p38 MAP kinases in myoblasts.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.

Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Models.

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK ß1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK ß1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Chemoproteomic analysis of intertissue and interspecies isoform diversity of AMP-activated protein kinase (AMPK).

AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that senses and governs changes in the cellular energy balance represented by concentrations of AMP, ADP, and ATP. Each of its three chains (α, ß, and γ) exists as either two or three subtypes, theoretically allowing up to 12 different forms of the complete enzyme. Tissue specificity in the distribution of AMPK subtypes is believed to underpin a range of biological functions for AMPK, a central regulator of metabolic function and response. It is of particular interest for drug discovery purposes to compare AMPK isoforms that are most prevalent in human liver and muscle with isoforms present in key preclinical species. To complement immunocapture/immunodetection methods, which for AMPK are challenged by sequence similarities and difficulties of obtaining accurate relative quantitation, AMPK was captured from lysates of a range of cells and tissues using the ActivX ATP probe. This chemical probe covalently attaches desthiobiotin to one or more conserved lysyl residues in the ATP-binding sites of protein kinases, including AMPK, while also labeling a wide range of ATP-utilizing proteins. Affinity-based recovery of labeled proteins followed by gel-based fractionation of the captured sample was followed by proteomic characterization of AMPK polypeptides. In agreement with transcript-based analysis and previous indications from immunodetection, the results indicated that the predominant AMPK heterotrimer in human liver is α1ß2γ1 but that dog and rat livers mainly contain the α1ß1γ1 and α2ß1γ1 forms, respectively. Differences were not detected between the AMPK profiles of normal and diabetic human liver tissues.

Large-scale phosphosite quantification in tissues by a spike-in SILAC method.

Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.

DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation.

Diet-induced obesity (DIO) leads to inflammatory activation of macrophages in white adipose tissue (WAT) and subsequently to insulin resistance. PPARgamma agonists are antidiabetic agents known to suppress inflammatory macrophage activation and to induce expression of the triacylglycerol (TG) synthesis enzyme acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) in WAT and in adipocytes. Here, we investigated in mice the relationship between macrophage lipid storage capacity and DIO-associated inflammatory macrophage activation. Mice overexpressing DGAT1 in both macrophages and adipocytes (referred to herein as aP2-Dgat1 mice) were more prone to DIO but were protected against inflammatory macrophage activation, macrophage accumulation in WAT, systemic inflammation, and insulin resistance. To assess the contribution of macrophage DGAT1 expression to this phenotype, we transplanted wild-type mice with aP2-Dgat1 BM. These mice developed DIO similar to that of control mice but retained the protection from WAT inflammation and insulin resistance seen in aP2-Dgat1 mice. In isolated macrophages, Dgat1 mRNA levels correlated directly with TG storage capacity and inversely with inflammatory activation by saturated fatty acids (FAs). Moreover, PPARgamma agonists increased macrophage Dgat1 mRNA levels, and the protective effects of these agonists against FA-induced inflammatory macrophage activation were absent in macrophages isolated from Dgat1-null mice. Thus, increasing DGAT1 expression in murine macrophages increases their capacity for TG storage, protects against FA-induced inflammatory activation, and is sufficient to reduce the inflammatory and metabolic consequences of DIO.

Quantitative proteomic analysis of single pancreatic islets.

Technological developments make mass spectrometry (MS)-based proteomics a central pillar of biochemical research. MS has been very successful in cell culture systems, where sample amounts are not limiting. To extend its capabilities to extremely small, physiologically distinct cell types isolated from tissue, we developed a high sensitivity chromatographic system that measures nanogram protein mixtures for 8 h with very high resolution. This technology is based on splitting gradient effluents into a capture capillary and provides an inherent technical replicate. In a single analysis, this allowed us to characterize kidney glomeruli isolated by laser capture microdissection to a depth of more than 2,400 proteins. From pooled pancreatic islets of Langerhans, another type of "miniorgan," we obtained an in-depth proteome of 6,873 proteins, many of them involved in diabetes. We quantitatively compared the proteome of single islets, containing 2,000-4,000 cells, treated with high or low glucose levels, and covered most of the characteristic functions of beta cells. Our ultrasensitive analysis recapitulated known hyperglycemic changes but we also find components up-regulated such as the mitochondrial stress regulator Park7. Direct proteomic analysis of functionally distinct cellular structures opens up perspectives in physiology and pathology.

Specific role for acyl CoA:Diacylglycerol acyltransferase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids.

UNLABELLED: Nonalcoholic fatty liver disease, characterized by the accumulation of triacylglycerols (TGs) and other lipids in the liver, often accompanies obesity and is a risk factor for nonalcoholic steatohepatitis and fibrosis. To treat or prevent fatty liver, a thorough understanding of hepatic fatty acid and TG metabolism is crucial. To investigate the role of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of TG synthesis, in fatty liver development, we studied mice with global and liver-specific knockout of Dgat1. DGAT1 was required for hepatic steatosis induced by a high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids. In contrast, lipodystrophy (aP2-SREBP-1c436) and liver X receptor activation (T0901317), which increase de novo fatty acid synthesis in liver, caused steatosis independently of DGAT1. Pharmacologic inhibition of Dgat1 with antisense oligonucleotides protected against fatty liver induced by a high-fat diet. CONCLUSION: Our findings identify a specific role for hepatic DGAT1 in esterification of exogenous fatty acids and indicate that DGAT1 contributes to hepatic steatosis induced by this mechanism.

Increased lipid accumulation and insulin resistance in transgenic mice expressing DGAT2 in glycolytic (type II) muscle.

Insulin resistance and type 2 diabetes are frequently accompanied by lipid accumulation in skeletal muscle. However, it is unknown whether primary lipid deposition in skeletal muscle is sufficient to cause insulin resistance or whether the type of muscle fiber, oxidative or glycolytic fiber, is an important determinant of lipid-mediated insulin resistance. Here we utilized transgenic mice to test the hypothesis that lipid accumulation specifically in glycolytic muscle promotes insulin resistance. Overexpression of DGAT2, which encodes an acyl-CoA:diacylglycerol acyltransferase that catalyzes triacylglycerol (TG) synthesis, in glycolytic muscle of mice increased the content of TG, ceramides, and unsaturated long-chain fatty acyl-CoAs in young adult mice. This lipid accumulation was accompanied by impaired insulin signaling and insulin-mediated glucose uptake in glycolytic muscle and impaired whole body glucose and insulin tolerance. We conclude that DGAT2-mediated lipid deposition specifically in glycolytic muscle promotes insulin resistance in this tissue and may contribute to the development of diabetes.

Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver.

Hepatic steatosis, the accumulation of lipids in the liver, is widely believed to result in insulin resistance. To test the causal relationship between hepatic steatosis and insulin resistance, we generated mice that overexpress acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step of triacylglycerol (TG) biosynthesis, in the liver (Liv-DGAT2 mice). Liv-DGAT2 mice developed hepatic steatosis, with increased amounts of TG, diacylglycerol, ceramides, and unsaturated long-chain fatty acyl-CoAs in the liver. However, they had no abnormalities in plasma glucose and insulin levels, glucose and insulin tolerance, rates of glucose infusion and hepatic glucose production during hyperinsulinemic-euglycemic clamp studies, or activities of insulin-stimulated signaling proteins in the liver. DGAT1 overexpression in the liver also failed to induce glucose or insulin intolerance. Our results indicate that DGAT-mediated lipid accumulation in the liver is insufficient to cause insulin resistance and show that hepatic steatosis can occur independently of insulin resistance.

Raloxifene inhibits matrix metalloproteinases expression and activity in macrophages and smooth muscle cells.

Secretion of matrix metalloproteinases (MMPs) by macrophages and smooth muscle cells (SMC) may impair atherosclerotic cap integrity leading to atherosclerosis complications. Selective estrogen receptor modulators (SERMs) have favourable impact on plasma lipid levels, but their role in the prevention of atherosclerosis still remains unclear. We investigated the effects of raloxifene, a second generation SERM, on MMP expression and activity in cultured macrophages and SMC, and in rabbit carotid lesions. Human monocyte-derived macrophages were isolated from blood of healthy donors. SMC were isolated from the intima-media layers of collared rabbit carotid arteries. Cells were incubated for 24h with increasing concentrations of raloxifene. Ovariectomized rabbits fed a 1% cholesterol-rich diet were subjected to pericarotid collar placement and treated with or without 10mgkg(-1)d(-1) raloxifene for 2 weeks. In macrophages, raloxifene treatment (0.1-10microM) significantly reduced MMP-9 gelatinolytic potential in a concentration-dependent manner, without affecting MMP-9 activation. This effect was estrogen receptor (ER)-dependent and due to the inhibition of MMP-9 promoter-driven transcription following an interaction with NF-kB pathway. Similarly, in cultured SMC, raloxifene inhibited up to 40% MMP-2 gelatinolytic activity. In vivo, raloxifene decreased the expression of MMP-2, MMP-3, and MMP-9 by intimal cells and the total gelatinolytic activity of collared carotids. These effects were accompanied by reduction of lesion size and inhibition of macrophage accumulation. Overall, results indicate that raloxifene may reduce MMPs expression and activity in macrophages and smooth muscle cells and favourably affect lesion formation.

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection.

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.