Complex Proteomes Research Articles

On the evolution of chaperones and cochaperones and the expansion of proteomes across the Tree of Life

Across the Tree of Life (ToL), the complexity of proteomes varies widely. Our systematic analysis depicts that from the simplest archaea to mammals, the total number of proteins per proteome expanded ∼200-fold. Individual proteins also became larger, and multidomain proteins expanded ∼50-fold. Apart from duplication and divergence of existing proteins, completely new proteins were born. Along the ToL, the number of different folds expanded ∼5-fold and fold combinations ∼20-fold. Proteins prone to misfolding and aggregation, such as repeat and beta-rich proteins, proliferated ∼600-fold and, accordingly, proteins predicted as aggregation-prone became 6-fold more frequent in mammalian compared with bacterial proteomes. To control the quality of these expanding proteomes, core chaperones, ranging from heat shock proteins 20 (HSP20s) that prevent aggregation to HSP60, HSP70, HSP90, and HSP100 acting as adenosine triphosphate (ATP)-fueled unfolding and refolding machines, also evolved. However, these core chaperones were already available in prokaryotes, and they comprise ∼0.3% of all genes from archaea to mammals. This challenge-roughly the same number of core chaperones supporting a massive expansion of proteomes-was met by 1) elevation of messenger RNA (mRNA) and protein abundances of the ancient generalist core chaperones in the cell, and 2) continuous emergence of new substrate-binding and nucleotide-exchange factor cochaperones that function cooperatively with core chaperones as a network.

Novel Strategies to Address the Challenges in Top-Down Proteomics.

Top-down mass spectrometry (MS)-based proteomics is a powerful technology for comprehensively characterizing proteoforms to decipher post-translational modifications (PTMs) together with genetic variations and alternative splicing isoforms toward a proteome-wide understanding of protein functions. In the past decade, top-down proteomics has experienced rapid growth benefiting from groundbreaking technological advances, which have begun to reveal the potential of top-down proteomics for understanding basic biological functions, unraveling disease mechanisms, and discovering new biomarkers. However, many challenges remain to be comprehensively addressed. In this Account & Perspective, we discuss the major challenges currently facing the top-down proteomics field, particularly in protein solubility, proteome dynamic range, proteome complexity, data analysis, proteoform-function relationship, and analytical throughput for precision medicine. We specifically review the major technology developments addressing these challenges with an emphasis on our research group's efforts, including the development of top-down MS-compatible surfactants for protein solubilization, functionalized nanoparticles for the enrichment of low-abundance proteoforms, strategies for multidimensional chromatography separation of proteins, and a new comprehensive user-friendly software package for top-down proteomics. We have also made efforts to connect proteoforms with biological functions and provide our visions on what the future holds for top-down proteomics.

Peptidyl Activity-Based Probes for Imaging Serine Proteases.

Proteases catalyze the hydrolysis of peptide bonds. Products of this breakdown mediate signaling in an enormous number of biological processes. Serine proteases constitute the most numerous group of proteases, accounting for 40%, and they are prevalent in many physiological functions, both normal and disease-related functions, making them one of the most important enzymes in humans. The activity of proteases is controlled at the expression level by posttranslational modifications and/or endogenous inhibitors. The study of serine proteases requires specific reagents not only for detecting their activity but also for their imaging. Such tools include inhibitors or substrate-related chemical molecules that allow the detection of proteolysis and visual observation of active enzymes, thus facilitating the characterization of the activity of proteases in the complex proteome. Peptidyl activity-based probes (ABPs) have been extensively studied recently, and this review describes the basic principles in the design of peptide-based imaging agents for serine proteases, provides examples of activity-based probe applications and critically discusses their strengths, weaknesses, challenges and limitations.

Secretome Profiling and Computational Biology of Human Leiomyoma Samples Unravel Molecular Signatures with Potential for Diagnostic and Therapeutic Interventions.

In recent years, significant advancements have been made in the way the complex proteome samples are compared but the ultimate goal of routine biomarker discovery has yet to be achieved. Based on reverse genetic strategy, our study involved the spotting of genes showing expressional variability in uterine leiomyoma females. Serum samples were taken from uterine leiomyomas subjects (n=6) and healthy control subjects (n=6) for proteomic studies. Additionally, leiomyoma tissue samples (n=25) and normal myometrium samples (n=25) were taken for validation studies. In this study, we profiled the proteomes of uterine leiomyoma patient's serum and healthy control, along with relative quantification using Nano LC-MS/MS analysis. A total of 146 proteins were reported to be significantly differentially expressed (P value less than 0.05) in case and control sample. Statistical analysis identified a number of molecular signatures distinguishing healthy from diseased serum. Among these, five proteins lumican, ficolin, MASP2, EMSY, and kallistatin were further chosen according to their function for validation. Kallistatin was downregulated while ficolin, MASP2, lumican, and EMSY were found to be upregulated in the diseased sample. The expression modulations in the identified genes were further validated in twenty-five cases. Interactions among the differentially expressed proteins were identified followed with network analysis. Network analysis emphasized important pathways that are highly deregulated in myoma, and functional significance of these pathways in the pathology of the disease was discussed. Comparative expression analysis reveals distinct molecular signatures and their probable role in diagnosis of the disease.

A tri-functional amino acid enables mapping of binding sites for posttranslational-modification-mediated protein-protein interactions

Posttranslational modification (PTM), through the recruitment of effector proteins (i.e., "readers") that signal downstream events, plays key roles in regulating a variety of cellular processes. To understand how a PTM is recognized, it is necessary to find its readers and, importantly, the location of the binding pockets responsible for PTM recognition. Although various methods have been developed to identify PTM readers, it remains a challenge to directly map the PTM-binding regions, especially for intrinsically disordered domains. Here, we demonstrate a photo-crosslinkable, clickable, and cleavable tri-functional amino acid, ADdis-Cys, that when coupled with mass spectrometry (ADdis-Cys-MS) can not only identify PTM readers from complex proteomes but also simultaneously map their PTM-recognition modules. Using ADdis-Cys-MS, we successfully identify the binding sites of several reader-PTM interactions, among which we discover human C1QBP as a histone chaperone. This robust method should find wide applications in examining other histone or non-histone PTM-mediated protein-protein interactions.

A Mini Review on Capillary Isoelectric Focusing-Mass Spectrometry for Top-Down Proteomics.

Mass spectrometry (MS)-based top-down proteomics (TDP) requires high-resolution separation of proteoforms before electrospray ionization (ESI)-MS and tandem mass spectrometry (MS/MS). Capillary isoelectric focusing (cIEF)-ESI-MS and MS/MS could be an ideal method for TDP because cIEF can enable separation of proteoforms based on their isoelectric points (pIs) with ultra-high resolution. cIEF-ESI-MS has been well-recognized for protein characterization since 1990s. However, the widespread adoption of cIEF-MS for the characterization of proteoforms had been impeded by several technical challenges, including the lack of highly sensitive and robust ESI interface for coupling cIEF to MS, ESI suppression of analytes from ampholytes, and the requirement of manual operations. In this mini review, we summarize the technical improvements of cIEF-ESI-MS for characterizing proteoforms and highlight some recent applications to hydrophobic proteins, urinary albumin variants, charge variants of monoclonal antibodies, and large-scale TDP of complex proteomes.

Iron Deficiency and Recovery in Yeast: A Quantitative Proteomics Approach.

Iron is an essential element for life, as it is critical for oxygen transport, cellular respiration, DNA synthesis, and metabolism. Disruptions in iron metabolism have been associated with several complex diseases like diabetes, cancer, infection susceptibility, neurodegeneration, and others; however, the molecular mechanisms linking iron metabolism with these diseases are not fully understood. A commonly used model to study iron deficiency (ID) is yeast, Saccharomyces cerevisiae. Here, we used quantitative (phospho)proteomics to explore the early (4 and 6 h) and late (12 h) response to ID. We showed that metabolic pathways like the Krebs cycle, amino acid, and ergosterol biosynthesis were affected by ID. In addition, during the late response, several proteins related to the ubiquitin-proteasome system and autophagy were upregulated. We also explored the proteomic changes during a recovery period after 12 h of ID. Several proteins recovered their steady-state levels, but some others, such as cytochromes, did not recover during the time tested. Additionally, we showed that autophagy is active during ID, and some of the degraded proteins during ID can be rescued using KO strains for several key autophagy genes. Our results highlight the complex proteome changes occurring during ID and recovery. This study constitutes a valuable data set for researchers interested in iron biology, offering a temporal proteomic data set for ID, as well as a compendium the proteomic changes associated with episodes of iron recovery.

Posttranslational modifications of the cytoskeleton.

The cytoskeleton plays important roles in many essential processes at the cellular and organismal levels, including cell migration and motility, cell division, and the establishment and maintenance of cell and tissue architecture. In order to facilitate these varied functions, the main cytoskeletal components-microtubules, actin filaments, and intermediate filaments-must form highly diverse intracellular arrays in different subcellular areas and cell types. The question of how this diversity is conferred has been the focus of research for decades. One key mechanism is the addition of posttranslational modifications (PTMs) to the major cytoskeletal proteins. This posttranslational addition of various chemical groups dramatically increases the complexity of the cytoskeletal proteome and helps facilitate major global and local cytoskeletal functions. Cytoskeletal proteins undergo many PTMs, most of which are not well understood. Recent technological advances in proteomics and cell biology have allowed for the in-depth study of individual PTMs and their functions in the cytoskeleton. Here, we provide an overview of the major PTMs that occur on the main structural components of the three cytoskeletal systems-tubulin, actin, and intermediate filament proteins-and highlight the cellular function of these modifications.

Exploring the diversity of plant proteome.

The tremendous functional, spatial, and temporal diversity of the plant proteome is regulated by multiple factors that continuously modify protein abundance, modifications, interactions, localization, and activity to meet the dynamic needs of plants. Dissecting the proteome complexity and its underlying genetic variation is attracting increasing research attention. Mass spectrometry (MS)-based proteomics has become a powerful approach in the global study of protein functions and their relationships on a systems level. Here, we review recent breakthroughs and strategies adopted to unravel the diversity of the proteome, with a specific focus on the methods used to analyze posttranslational modifications (PTMs), protein localization, and the organization of proteins into functional modules. We also consider PTM crosstalk and multiple PTMs temporally regulating the life cycle of proteins. Finally, we discuss recent quantitative studies using MS to measure protein turnover rates and examine future directions in the study of the plant proteome.

Methodological considerations for identifying multiple plasma proteins associated with all-cause mortality in a population-based prospective cohort

Novel methods to characterize the plasma proteome has made it possible to examine a wide range of proteins in large longitudinal cohort studies, but the complexity of the human proteome makes it difficult to identify robust protein-disease associations. Nevertheless, identification of individuals at high risk of early mortality is a central issue in clinical decision making and novel biomarkers may be useful to improve risk stratification. With adjustment for established risk factors, we examined the associations between 138 plasma proteins measured using two proximity extension assays and long-term risk of all-cause mortality in 3,918 participants of the population-based Malmö Diet and Cancer Study. To examine the reproducibility of protein-mortality associations we used a two-step random-split approach to simulate a discovery and replication cohort and conducted analyses using four different methods: Cox regression, stepwise Cox regression, Lasso-Cox regression, and random survival forest (RSF). In the total study population, we identified eight proteins that associated with all-cause mortality after adjustment for established risk factors and with Bonferroni correction for multiple testing. In the two-step analyses, the number of proteins selected for model inclusion in both random samples ranged from 6 to 21 depending on the method used. However, only three proteins were consistently included in both samples across all four methods (growth/differentiation factor-15 (GDF-15), N-terminal pro-B-type natriuretic peptide, and epididymal secretory protein E4). Using the total study population, the C-statistic for a model including established risk factors was 0.7222 and increased to 0.7284 with inclusion of the most predictive protein (GDF-15; P < 0.0001). All multiple protein models showed additional improvement in the C-statistic compared to the single protein model (all P < 0.0001). We identified several plasma proteins associated with increased risk of all-cause mortality independently of established risk factors. Further investigation into the putatively causal role of these proteins for longevity is needed. In addition, the examined methods for identifying multiple proteins showed tendencies for overfitting by including several putatively false positive findings. Thus, the reproducibility of findings using such approaches may be limited.

Genetic disruption of WASHC4 drives endo-lysosomal dysfunction and cognitive-movement impairments in mice and humans.

Mutation of the Wiskott-Aldrich syndrome protein and SCAR homology (WASH) complex subunit, SWIP, is implicated in human intellectual disability, but the cellular etiology of this association is unknown. We identify the neuronal WASH complex proteome, revealing a network of endosomal proteins. To uncover how dysfunction of endosomal SWIP leads to disease, we generate a mouse model of the human WASHC4c.3056C>G mutation. Quantitative spatial proteomics analysis of SWIPP1019R mouse brain reveals that this mutation destabilizes the WASH complex and uncovers significant perturbations in both endosomal and lysosomal pathways. Cellular and histological analyses confirm that SWIPP1019R results in endo-lysosomal disruption and uncover indicators of neurodegeneration. We find that SWIPP1019R not only impacts cognition, but also causes significant progressive motor deficits in mice. A retrospective analysis of SWIPP1019R patients reveals similar movement deficits in humans. Combined, these findings support the model that WASH complex destabilization, resulting from SWIPP1019R, drives cognitive and motor impairments via endo-lysosomal dysfunction in the brain.

Application of frequency multiple FT-ICR MS signal acquisition for improved proteome research

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with liquid chromatography (LC) is a powerful combination useful in many research areas due to the utility of high mass resolving power and mass measurement accuracy for studying highly complex samples. Ideally, every analyte in a complex sample can be subjected to accurate mass MS/MS analysis to aid in identification. FT-ICR MS can provide high mass resolving power and mass accuracy at the cost of long data acquisition periods, reducing the number of spectra that can be acquired per unit time. Frequency multiple signal acquisition has long been realized as an attractive method to obtain high mass resolving power and mass accuracy with shorter data acquisition periods. However, one of the limitations associated with frequency multiple signal acquisition is reduced signal intensity as compared to a traditional dipole detector. In this study, we demonstrated the use of a novel ICR cell to improve frequency multiple signal intensity and investigated the potential use of frequency multiple acquisition for proteome measurements. This novel ICR cell containing both dipole and frequency multiple detection electrodes was installed on a 7T FT-ICR MS coupled to an LC system. Tryptic digests of HeLa cell lysates were analyzed using dipole and frequency multiple detectors by holding either the mass resolving power or signal acquisition time constant. Compared to dipole detection, second frequency multiple detection yielded 36% or 45% more unique identified peptides from HeLa cell lysates at twice the scan rate or twice the mass resolving power, respectively. These results indicate that frequency multiple signal acquisition with either the same resolving power or the same signal acquisition duration as used with dipole signals can produce a significant increase in the number of peptides identified in complex proteome samples.

ProteaseGuru: A Tool for Protease Selection in Bottom-Up Proteomics.

Bottom-up proteomics is currently the dominant strategy for proteome analysis. It relies critically upon the use of a protease to digest proteins into peptides, which are then identified by liquid chromatography-mass spectrometry (LC-MS). The choice of protease(s) has a substantial impact upon the utility of the bottom-up results obtained. Protease selection determines the nature of the peptides produced, which in turn affects the ability to infer the presence and quantities of the parent proteins and post-translational modifications in the sample. We present here the software tool ProteaseGuru, which provides in silico digestions by candidate proteases, allowing evaluation of their utility for bottom-up proteomic experiments. This information is useful for both studies focused on a single or small number of proteins, and for analysis of entire complex proteomes. ProteaseGuru provides a convenient user interface, valuable peptide information, and data visualizations enabling the comparison of digestion results of different proteases. The information provided includes data tables of theoretical peptide sequences and their biophysical properties, results summaries outlining the numbers of shared and unique peptides per protease, histograms facilitating the comparison of proteome-wide proteolytic data, protein-specific summaries, and sequence coverage maps. Examples are provided of its use to inform analysis of variant-containing proteins in the human proteome, as well as for studies requiring the use of multiple proteomic databases such as a human:mouse xenograft model, and microbiome metaproteomics.

Role of promoters in regulating alternative splicing.

Alternative splicing (AS) plays a critical role in enhancing proteome complexity in higher eukaryotes. Almost all the multi intron-containing genes undergo AS in humans. Splicing mainly occurs co-transcriptionally, where RNA polymerase II (RNA pol II) plays a crucial role in coordinating transcription and pre-mRNA splicing. Aberrant AS leads to non-functional proteins causative in various pathophysiological conditions such as cancers, neurodegenerative diseases, and muscular dystrophies. Transcription and pre-mRNA splicing are deeply interconnected and can influence each other's functions. Several studies evinced that specific promoters employed by RNA pol II dictate the RNA processing decisions. Promoter-specific recruitment of certain transcriptional factors or transcriptional coactivators influences splicing, and the extent to which these factors affect splicing has not been discussed in detail. Here, in this review, various DNA-binding proteins and their influence on promoter-specific AS are extensively discussed. Besides, this review highlights how the promoter-specific epigenetic changes might regulate AS.

Most non-canonical proteins uniquely populate the proteome or immunopeptidome

Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability, and critically generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5' "untranslated" regions hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.

Comparing the Use of Magnetic Beads with Ultrafiltration for Ancient Dental Calculus Proteomics.

Over the past two decades, proteomic analysis has greatly developed in application to the field of biomolecular archaeology, coinciding with advancements in LC-MS/MS instrumentation sensitivity and improvements in sample preparation methods. Recently, human dental calculus has received much attention for its well-preserved proteomes locked in mineralized dental plaque which stores information on human diets and the oral microbiome otherwise invisible to other biomolecular approaches. Maximizing proteome recovery in ancient dental calculus, available only in minute quantities and irreplaceable after destructive analysis, is of paramount importance. Here, we compare the more traditional ultrafiltration-based and acetone precipitation approaches with the newer paramagnetic bead approach in order to test the influence of demineralization acid on recovered proteome complexity obtained from specimens as well as the sequence coverages matched for significant proteins. We found that a protocol utilizing EDTA combined with paramagnetic beads increased proteome complexity, in some cases doubling the number of unique peptides and number of proteins matched, compared to protocols involving the use of HCl and either acetone precipitation or ultrafiltration. Although the increase in the number of proteins was almost exclusively of bacterial origin, a development that has implications for the study of diseases within these ancient populations, an increase in the peptide number for the dairy proteins β-lactoglobulin and casein was also observed reflecting an increase in sequence coverage for these dietary proteins of interest. We also consider structural explanations for the discrepancies observed between these two key dietary proteins preserved in archaeological dental calculus.

Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques.

Human plasma is one of the most widely used tissues in clinical analysis, and plasma-based biomarkers are used for monitoring patient health status and/or response to medical treatment to avoid unnecessary invasive biopsy. Data-driven plasma proteomics has suffered from a lack of throughput and detection sensitivity, largely due to the complexity of the plasma proteome and in particular the enormous quantitative dynamic range, estimated to be between 9 and 13 orders of magnitude between the lowest and the highest abundance protein. A major challenge is to identify workflows that can achieve depth of plasma proteome coverage while minimizing the complexity of the sample workup and maximizing the sample throughput. In this study, we have performed intensive depletion of high-abundant plasma proteins or enrichment of low-abundant proteins using the Agilent multiple affinity removal liquid chromatography (LC) column-Human 6 (Hu6), the Agilent multiple affinity removal LC column-Human 14 (Hu14), and ProteoMiner followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and C18 prefractionation techniques. We compared the performance of each of these fractionation approaches to identify the method that satisfies requirements for analysis of clinical samples and to include good plasma proteome coverage in combination with reasonable sample output. In this study, we report that one-dimensional (1D) gel-based prefractionation allows parallel sample processing and no loss of proteome coverage, compared with serial chromatographic separation, and significantly accelerates analysis time, particularly important for large clinical projects. Furthermore, we show that a variety of methodologies can achieve similarly high plasma proteome coverage, allowing flexibility in method selection based on project-specific needs. These considerations are important in the effort to accelerate plasma proteomics research so as to provide efficient, reliable, and accurate diagnoses, population-based health screening, clinical research studies, and other clinical work.

Identification of differentially expressed proteins and clinicopathological significance of HMGB2 in cervical cancer

To investigate the complexity of proteomics in cervical cancer tissues, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry analysis on a panel of normal cervical tissues (N), high-grade squamous intraepithelial lesion tissues (HSIL) and cervical cancer tissues (CC). Total 72 differentially expressed proteins were identified both in CC vs N and CC vs HSIL. The expression of HMGB2 was markedly higher in CC than that in HSIL and N. High HMGB2 expression was significantly correlated with primary tumor size, invasion and tumor stage. The up-regulated HMGB2 was discovered to be associated with human cervical cancer. These findings suggest that HMGB2 may be a potentially prognostic biomarker and a target for the therapy of cervical cancer.

Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes.

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2β

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2β, is downregulated in models of muscle growth and that overexpression ASB2β is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2β expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2β-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2β overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2β knockdown induces mild muscle hypertrophy. ASB2β-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2β and, therefore, potential ASB2β targets, Flag-tagged wild-type ASB2β, and a mutant ASB2β lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2β interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2β target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction.