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Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein- C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2-/- mice showed that Itgb2 is required for proper WNT signaling regulation in the lung. © 2013 American Chemical Society.

Morphological and metabolic adaptations of the human skeletal muscle to exercise are crucial to improve performance and prevent chronic diseases and metabolic disorders. In this study we investigated human skeletal muscle protein composition in endurance trained (ET) versus untrained individuals (UT) and its modulation by an acute bout of endurance exercise. Participants were recruited based on their VO 2 max and subjected to a bicycle exercise test. M. vastus lateralis biopsies were taken before and three hours after exercise. Muscle lysates were analyzed using off-gel LC-MS/MS. Relative protein abundances were compared between ET and UT at rest and after exercise. Comparing UT and ET, we identified 92 significantly changed proteins under resting conditions. Specifically, fiber-type-specific and proteins of the oxidative phosphorylation and tricarboxylic acid cycle were increased in ET. In response to acute exercise, 71 proteins in ET and 44 in UT were altered. Here, a decrease of proteins involved in energy metabolism accompanied with alterations of heat shock and proteasomal proteins could be observed. In summary, long-term endurance training increased the basal level of structural and mitochondrial proteins in skeletal muscle. In contrast, acute exercise resulted in a depletion of proteins related to substrate utilization, especially in trained athletes. Biological significance The investigation of the human skeletal muscle proteome in response to exercise may provide novel insights into the process of muscular plasticity. It is of importance in the development of exercise-based strategies in the prevention and therapy of many chronic inflammatory and degenerative diseases which are often accompanied by muscular deconditioning. Up to date, proteomic investigations of the human muscle proteome in adaptation to exercise are mainly focused on untrained individuals and often restricted to animal studies. In the present study we compare the protein composition in endurance trained athletes and untrained individuals in the resting muscle and its modulation in response to acute exercise. To our knowledge, we present the first comprehensive analysis of skeletal muscle proteome alterations in response to acute and long-term exercise intervention.

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Purpose: Control of cardiomyocyte (CM) proteostasis operated by the ubiquitin/proteasome (UPS) and autophagy/lysosome systems is fundamental for heart adaptation to both physiologic and pathologic stresses. Reduced efficiency of either UPS or autophagy/lysosome system occurs during ageing and has been associated to cardiomyopathies. Atrogin-1 is a muscle specific ubiquitin ligase, targeting for degradation signalling proteins involved in cardiac hypertrophy. However, the role of Atrogin-1 in CM biology and its involvement in the molecular mechanism of cardiac dysfunction, are largely unexplored. Methods: We analyzed hearts from Atrogin-1 knock-out (KO) mice from 6 mo. onwards until death, and compared it to that of age- and sex-matched controls. Functional, immunofluorescence and electron microscopy analyses were performed. Markers of ER/SR stress and autophagy/lysosome systems were investigated by RTqPCR and WB. In vivo pulsed SILAC proteomics and bioinformatics, Co-IP, in vitro assays and in vivo viral silencing were performed to identify novel targets of Atrogin-1. Results: By using in vivo and in vitro assays we identified a novel target of Atrogin-1, the ESCRTIII protein CHMP2B, that plays a fundamental role in autophagy. Failure to degrade CHMP2B in Atrogin-1 KO mice caused autophagy impairment, accumulation of intracellular protein aggregates, activation of the unfolded protein response and subsequent CM apoptosis, all of which increased progressively during ageing. The alterations in cellular proteostasis resulted in cardiomyopathy with a restrictive pattern, characterized by myocardial remodelling with interstitial fibrosis, diastolic dysfunction (Edt, KO: 20.7±2.7 vs WT: 27.8±5.7, in msec), arrhythmias and secondary LA and ventricular remodelling, as well as CM hypertrophy (CM area, KO: 289.34±2.23 vs WT: 236.77±1.64, in μm2). Aged Atrogin-1 KO mice had reduced tolerance to treadmill exercise compared to controls, and shortened life span (KO: 17±1 vs WT: 24±1.2, in mo.). In vivo reduction of CHMP2B protein level in the KO mice restored normal autophagy and protected CMs from cell death resulting from CHMP2B proteotoxicity. Conclusions: Our data highlight the importance of regulated proteolysis in the heart and show that the loss of Atrogin-1 per se is sufficient to cause cardiac damage, which evolves into cardiomyopathy when protein quality control becomes less efficient, as occurring in aging. Such cardiomyopathy represents a novel model of proteotoxic myocardial remodelling and will be useful to determine the mechanism of impaired proteostasis to CM damage.