Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

Leveraging inter-individual transcriptional correlation structure to infer discrete signaling mechanisms across metabolic tissues.

Inter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. 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. 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. Expanding on this intuition, we reasoned that parallel strategies could be used to understand how individual genes mediate signaling across metabolic tissues through correlative analyses of gene variation between individuals. Thus, comparison of quantitative levels of gene expression relationships between organs in a population could aid in understanding cross-organ signaling. Here, we surveyed gene-gene correlation structure across 18 metabolic tissues in 310 human individuals and 7 tissues in 103 diverse strains of mice fed a normal chow or high-fat/high-sucrose (HFHS) diet. Variation of genes such as FGF21, ADIPOQ, GCG, and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both within-tissue signaling mechanisms (liver PCSK9) and genes encoding enzymes producing metabolites (adipose PNPLA2), where inter-individual correlation structure aligned with known roles for these critical metabolic pathways. Examination of sex hormone receptor correlations in mice highlighted the difference of tissue-specific variation in relationships with metabolic traits. We refer to this resource as gene-derived correlations across 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 correlated patterns of genes, cell types, pathways, and network architectures across metabolic organs.

Liver-derived extracellular vesicles improve whole-body glycaemic control via inter-organ communication.

Small extracellular vesicles (EVs) are signalling messengers that regulate inter-tissue communication through delivery of their molecular cargo. Here, we show that liver-derived EVs are acute regulators of whole-body glycaemic control in mice. Liver EV secretion into the circulation is increased in response to hyperglycaemia, resulting in increased glucose effectiveness and insulin secretion through direct inter-organ EV signalling to skeletal muscle and the pancreas, respectively. This acute blood glucose lowering effect occurs in healthy and obese mice with non-alcoholic fatty liver disease, despite marked remodelling of the liver-derived EV proteome in obese mice. The EV-mediated blood glucose lowering effects were recapitulated by administration of liver EVs derived from humans with or without progressive non-alcoholic fatty liver disease, suggesting broad functional conservation of liver EV signalling and potential therapeutic utility. Taken together, this work reveals a mechanism whereby liver EVs act on peripheral tissues via endocrine signalling to restore euglycaemia in the postprandial state.

SnapKin: a snapshot deep learning ensemble for kinase-substrate prediction from phosphoproteomics data.

A major challenge in mass spectrometry-based phosphoproteomics lies in identifying the substrates of kinases, as currently only a small fraction of substrates identified can be confidently linked with a known kinase. Machine learning techniques are promising approaches for leveraging large-scale phosphoproteomics data to computationally predict substrates of kinases. However, the small number of experimentally validated kinase substrates (true positive) and the high data noise in many phosphoproteomics datasets together limit their applicability and utility. Here, we aim to develop advanced kinase-substrate prediction methods to address these challenges. Using a collection of seven large phosphoproteomics datasets, and both traditional and deep learning models, we first demonstrate that a 'pseudo-positive' learning strategy for alleviating small sample size is effective at improving model predictive performance. We next show that a data resampling-based ensemble learning strategy is useful for improving model stability while further enhancing prediction. Lastly, we introduce an ensemble deep learning model ('SnapKin') by incorporating the above two learning strategies into a 'snapshot' ensemble learning algorithm. We propose SnapKin, an ensemble deep learning method, for predicting substrates of kinases from large-scale phosphoproteomics data. We demonstrate that SnapKin consistently outperforms existing methods in kinase-substrate prediction. SnapKin is freely available at https://github.com/PYangLab/SnapKin.

Impact of Bmal1 Rescue and Time-Restricted Feeding on Liver and Muscle Proteomes During the Active Phase in Mice.

Molecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding are well defined at the transcriptional level, their impact on governing protein abundance in peripheral tissues is unclear. Here, we determine the relative contributions of local molecular clocks and daily feeding cycles on liver and muscle proteomes during the active phase in mice. LC-MS/MS was performed on liver and gastrocnemius muscle harvested 4 h into the dark phase from WT, Bmal1 KO, and dual liver- and muscle-Bmal1-rescued mice under either ad libitum feeding or time-restricted feeding during the dark phase. Feeding-fasting cycles had only minimal effects on levels of liver proteins and few, if any, on the muscle proteome. In contrast, Bmal1 KO altered the abundance of 674 proteins in liver and 80 proteins in muscle. Local rescue of liver and muscle Bmal1 restored ∼50% of proteins in liver and ∼25% in muscle. These included proteins involved in fatty acid oxidation in liver and carbohydrate metabolism in muscle. For liver, proteins involved in de novo lipogenesis were largely dependent on Bmal1 function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 in liver and muscle were enriched for secreted proteins. We found that the abundance of fibroblast growth factor 1, a liver secreted protein, requires BMAL1 and that autocrine fibroblast growth factor 1 signaling modulates mitochondrial respiration in hepatocytes. In liver and muscle, BMAL1 is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and many metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis.

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.

GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

Liver and muscle circadian clocks cooperate to support glucose tolerance in mice.

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

Vascular cells improve functionality of human cardiac organoids.

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor ß (PDGFRß) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens.

Mass spectrometry-driven glycomics and glycoproteomics, the system-wide profiling of detached glycans and intact glycopeptides from biological samples, respectively, are powerful approaches to interrogate the heterogenous glycoproteome. Efforts to develop integrated workflows employing both glycomics and glycoproteomics have been invested since the concerted application of these complementary approaches enables a deeper exploration of the glycoproteome. This protocol paper outlines, step-by-step, an integrated -omics technology, the "glycomics-assisted glycoproteomics" method, that first establishes the N-glycan fine structures and their quantitative distribution pattern of protein extracts via porous graphitized carbon-LC-MS/MS. The N-glycome information is then used to augment and guide the challenging reversed-phase LC-MS/MS-based profiling of intact N-glycopeptides from the same protein samples. Experimental details and considerations relating to the sample preparation and the N-glycomics and N-glycoproteomics data collection, analysis, and integration are discussed. Benefits of the glycomics-assisted glycoproteomics method, which can be readily applied to both simple and complex biological specimens such as protein extracts from cells, tissues, and bodily fluids (e.g., serum), include quantitative information of the protein carriers and site(s) of glycosylation, site occupancy, and the site-specific glycan structures directly from biological samples. The glycomics-assisted glycoproteomics method therefore facilitates a comprehensive view of the complexity and dynamics of the heterogenous glycoproteome.

Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes.

Cancer cachexia is common in many cancers and the loss of skeletal muscle mass compromises the response to therapies and quality of life. A contributing mechanism is oxidative stress and compounds able to attenuate it may be protective. Sulforaphane (SFN), a natural antioxidant in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) signaling to decrease oxidative stress. Although SFN has potential as a cancer therapeutic, whether it can attenuate muscle wasting in the absence or presence of chemotherapy is unknown. In healthy C2C12 myotubes, SFN administration for 48 h induced hypertrophy through increased myoblast fusion via Nrf2 and ERK signaling. To determine whether SFN could attenuate wasting induced by cancer cells, myotubes were cocultured with or without Colon-26 (C-26) cancer cells for 48 h and treated with 5-fluorouracil (5-FU, 5 µM) or vehicle (DMSO). SFN (10 µM) or DMSO was added for the final 24 h. Coculture with cancer cells in the absence and presence of 5-FU reduced myotube width by ∼30% (P < 0.001) and ∼20% (P < 0.01), respectively, which was attenuated by SFN (P < 0.05). Exposure to C-26 conditioned media reduced myotube width by 15% (P < 0.001), which was attenuated by SFN. Western immunoblotting and qRT-PCR confirmed activation of Nrf2 signaling and antioxidant genes. Coadministration of Nrf2 inhibitors (ML-385) or MEK inhibitors (PD184352) revealed that SFN's attenuation of atrophy was blocked by ERK inhibition. These data support the chemoprotective and antioxidative function of SFN in myotubes, highlighting its therapeutic potential for cancer-related muscle wasting.

urPTMdb/TeaProt: Upstream and Downstream Proteomics Analysis.

We have developed the underrepresented post-translational modification (PTM) database (urPTMdb), a PTM gene set database to accelerate the discovery of enriched protein modifications in experimental data. urPTMdb provides curated lists of proteins reported to be substrates of underrepresented modifications. Their enrichment in proteomics datasets can reveal unexpected PTM regulations. urPTMdb can be implemented in existing workflows, or used in TeaProt, an online Shiny tool that integrates upstream transcription factor enrichment analysis with downstream pathway analysis through an easy-to-use interactive interface. TeaProt annotates user-uploaded data with drug-gene interactions, subcellular localizations, phenotypic functions, gene-disease associations, and enzyme-gene interactions. TeaProt enables gene set enrichment analysis (GSEA) to discover enrichments in gene sets from various resources, including MSigDB, CHEA, and urPTMdb. We demonstrate the utility of urPTMdb and TeaProt through the analysis of a previously published Western diet-induced remodeling of the tongue proteome, which revealed altered cellular processes associated with energy metabolism, interferon alpha/gamma response, adipogenesis, HMGylation substrate enrichment, and transcription regulation through PPARG and CEBPA. Additionally, we analyzed the interactome of ADP-ribose glycohydrolase TARG1, a key enzyme that removes mono-ADP-ribosylation. This analysis identified an enrichment of ADP-ribosylation, ribosomal proteins, and proteins localized in the nucleoli and endoplasmic reticulum. TeaProt and urPTMdb are accessible at https://tea.coffeeprot.com/.

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Focus on diet and exercise.

Research-based lifestyle choices can help us live healthier lives, and in this issue of Cell Metabolism, we showcase articles focused on diet and exercise interventions. Here, we take a moment to learn about the motivation and challenges behind these studies and look forward to the next steps in applying these interventions to promote metabolic health.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

Phosphoproteomics of three exercise modalities identifies canonical signaling and C18ORF25 as an AMPK substrate regulating skeletal muscle function.

Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.

Time-resolved phosphoproteome and proteome analysis reveals kinase signaling on master transcription factors during myogenesis.

Myogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.

SILAC kinase screen identifies potential MASTL substrates.

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.