RESUMEN
BACKGROUND: Extensive evidence from single-center studies indicates that a subset of patients with chronic advanced heart failure (HF) undergoing left ventricular assist device (LVAD) support show significantly improved heart function and reverse structural remodeling (ie, termed "responders"). Furthermore, we recently published a multicenter prospective study, RESTAGE-HF (Remission from Stage D Heart Failure), demonstrating that LVAD support combined with standard HF medications induced remarkable cardiac structural and functional improvement, leading to high rates of LVAD weaning and excellent long-term outcomes. This intriguing phenomenon provides great translational and clinical promise, although the underlying molecular mechanisms driving this recovery are largely unknown. METHODS: To identify changes in signaling pathways operative in the normal and failing human heart and to molecularly characterize patients who respond favorably to LVAD unloading, we performed global RNA sequencing and phosphopeptide profiling of left ventricular tissue from 93 patients with HF undergoing LVAD implantation (25 responders and 68 nonresponders) and 12 nonfailing donor hearts. Patients were prospectively monitored through echocardiography to characterize their myocardial structure and function and identify responders and nonresponders. RESULTS: These analyses identified 1341 transcripts and 288 phosphopeptides that are differentially regulated in cardiac tissue from nonfailing control samples and patients with HF. In addition, these unbiased molecular profiles identified a unique signature of 29 transcripts and 93 phosphopeptides in patients with HF that distinguished responders after LVAD unloading. Further analyses of these macromolecules highlighted differential regulation in 2 key pathways: cell cycle regulation and extracellular matrix/focal adhesions. CONCLUSIONS: This is the first study to characterize changes in the nonfailing and failing human heart by integrating multiple -omics platforms to identify molecular indices defining patients capable of myocardial recovery. These findings may guide patient selection for advanced HF therapies and identify new HF therapeutic targets.
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Insuficiencia Cardíaca , Trasplante de Corazón , Corazón Auxiliar , Humanos , Transcriptoma , Estudios Prospectivos , Fosfopéptidos/metabolismo , Proteómica , Donantes de Tejidos , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismoRESUMEN
Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker. Solvent accessible surface distance (SASD), which considers the accessibility of the cross-linked residues and the path connecting them, is a better predictor of consistency than the Euclidean distance. However, SASDs of structures are not publicly available, and their calculation is computationally intensive. Here, we describe in XLinkDB version 4.0 the automatic calculation of SASDs using Jwalk for all cross-links mapped to structures, both with and without regard to ligands, and derive empirical maximum SASD spans for BDP-NHP and DSSO cross-linkers of 51 and 43 Å, respectively. We document ligands proximal to cross-links in structures and demonstrate how SASDs can be used to help infer sample protein conformations and ligand occupancy, highlighting cross-links sensitive to ADP binding in mitochondria isolated from HEK293 cells.
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Proteínas , Reactivos de Enlaces Cruzados/química , Células HEK293 , Humanos , Ligandos , Conformación Proteica , Proteínas/químicaRESUMEN
Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.
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Epigénesis Genética , Miocardio/enzimología , Proteína Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Animales , Ensamble y Desensamble de Cromatina , Metilación de ADN , Cardiopatías/enzimología , Cardiopatías/genética , Humanos , Metilación , Ratones , Activación TranscripcionalRESUMEN
Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.
RESUMEN
Structural information on protein-protein interactions (PPIs) is essential for improved understanding of regulatory interactome networks that confer various physiological and pathological responses. Additionally, maladaptive PPIs constitute desirable therapeutic targets due to inherently high disease state specificity. Recent advances in chemical cross-linking strategies coupled with mass spectrometry (XL-MS) have positioned XL-MS as a promising technology to not only elucidate the molecular architecture of individual protein assemblies, but also to characterize proteome-wide PPI networks. Moreover, quantitative in vivo XL-MS provides a new capability for the visualization of cellular interactome dynamics elicited by drug treatments, disease states, or aging effects. The emerging field of XL-MS based complexomics enables unique insights on protein moonlighting and protein complex remodeling. These techniques provide complimentary information necessary for in-depth structural interactome studies to better comprehend how PPIs mediate function in living systems.
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Reactivos de Enlaces Cruzados , Espectrometría de Masas , Reactivos de Enlaces Cruzados/química , Espectrometría de Masas/métodos , Humanos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Biología de Sistemas/métodos , Mapas de Interacción de Proteínas , Animales , Proteómica/métodosRESUMEN
Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid ß-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young-old animal pairs. Each observed cross-link can serve as a protein conformational or protein-protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.
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Mitocondrias , Proteómica , Humanos , Ratones , Femenino , Animales , Anciano , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Envejecimiento/metabolismo , Complejo I de Transporte de Electrón/metabolismoRESUMEN
Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.
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Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.
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Canales de Calcio , Calcio , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Homeostasis , Mitocondrias/metabolismoRESUMEN
In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.