RESUMEN
Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.
Asunto(s)
Fosfopéptidos , Proteínas Proto-Oncogénicas c-akt , Fosforilación , Fosfopéptidos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Fosfoproteínas/metabolismoRESUMEN
Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.
Asunto(s)
Enfermedad de Alexander , Enfermedad de Alexander/genética , Enfermedad de Alexander/metabolismo , Enfermedad de Alexander/patología , Animales , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Gliosis/metabolismo , Gliosis/patología , Humanos , Ratones , Ratones Transgénicos , Mutación , ProteómicaRESUMEN
The cardiac isoform of troponin I has a unique N-terminal extension (~ 1-30 amino acids), which contributes to the modulation of cardiac contraction and relaxation. Hearts of various species including humans produce a truncated variant of cardiac troponin I (cTnI-ND) deleting the first ~ 30 amino acids as an adaption in pathophysiological conditions. In this study, we investigated the impact of cTnI-ND chronic expression in transgenic mouse hearts compared to wildtype (WT) controls (biological n = 8 in each group). We aimed to determine the global phosphorylation effects of cTnI-ND on the cardiac proteome, thereby determining the signaling pathways that have an impact on cardiac function. The samples were digested and isobarically labeled and equally mixed for relative quantification via nanoLC-MS/MS. The peptides were then enriched for phospho-peptides and bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA). We found approximately 77% replacement of the endogenous intact cTnI with cTnI-ND in the transgenic mouse hearts with 1674 phospho-proteins and 2971 non-modified proteins. There were 73 significantly altered phospho-proteins; bioinformatic analysis identified the top canonical pathways as associated with integrin, protein kinase A, RhoA, and actin cytoskeleton signaling. Among the 73 phospho-proteins compared to controls cTnI-ND hearts demonstrated a significant decrease in paxillin and YAP1, which are known to play a role in cell mechano-sensing pathways. Our data indicate that cTnI-ND modifications in the sarcomere are sufficient to initiate changes in the phospho-signaling profile that may underly the chronic-adaptive response associated with cTnI cleavage in response to stressors by modifying mechano-sensitive signaling pathways.
Asunto(s)
Espectrometría de Masas en Tándem , Troponina I , Aminoácidos , Animales , Calcio/metabolismo , Ratones , Ratones Transgénicos , Contracción Miocárdica , Miocardio/metabolismo , Péptidos , Fosforilación , Transducción de Señal , Troponina I/química , Troponina I/genética , Troponina I/metabolismoRESUMEN
RATIONALE: CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. OBJECTIVE: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. METHODS AND RESULTS: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. CONCLUSIONS: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Cardiomegalia/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Aorta , Arritmias Cardíacas/etiología , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Estimulación Cardíaca Artificial , Cardiomegalia/patología , Núcleo Celular/metabolismo , Constricción , Citosol/metabolismo , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Histona Desacetilasas/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/citología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factores de Tiempo , Activación TranscripcionalRESUMEN
Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Cromatografía Liquida , Genotipo , Humanos , Proteínas/genéticaRESUMEN
The protein p62/Sequestosome 1 (p62) has been described as a selective autophagy receptor and independently as a platform for pro-inflammatory and other intracellular signaling. How these seemingly disparate functional roles of p62 are coordinated has not been resolved. Here, we show that TAK1, a kinase involved in immune signaling, negatively regulates p62 action in autophagy. TAK1 reduces p62 localization to autophagosomes, dampening the autophagic degradation of both p62 and p62-directed autophagy substrates. TAK1 also relocalizes p62 into dynamic cytoplasmic bodies, a phenomenon that accompanies the stabilization of TAK1 complex components. On the other hand, p62 facilitates the assembly and activation of TAK1 complexes, suggesting a connection between p62's signaling functions and p62 body formation. Thus, TAK1 governs p62 action, switching it from an autophagy receptor to a signaling platform. This ability of TAK1 to disable p62 as an autophagy receptor may allow certain autophagic substrates to accumulate when needed for cellular functions.
Asunto(s)
Autofagia/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/metabolismo , Autofagosomas/metabolismo , Autofagia/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Quinasas Quinasa Quinasa PAM/genética , Microscopía Confocal , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteína Sequestosoma-1/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Milk fat globule membrane (MFGM) is a glycosylated, protein-embedded, phospholipid fraction that surrounds triglycerides in milk. Commercial bovine sources have recently come to the market as a novel food ingredient and have been added to various products, including infant formula. Considering that MFGM is a heterogeneous mixture of fat, protein, and carbohydrate, it can be expected that variations among MFGM products exist. For this reason, our aim was to characterize the composition of commercial MFGM samples through a combination of proteomic and lipidomic analyses. Six bovine milk fractions, represented as MFGM fractions or phospholipid fractions, were obtained from various commercial sources. Additionally, the MFGM samples were compared with 2 infant formulas, a standard formula as well as a premium formula containing MFGM. For proteomic analysis, bottom-up data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on each MFGM fraction, and nearly a thousand proteins were identified across all samples, with 364 of them having different abundance across the samples tested. One hundred twelve proteins differed by a fold-change of 10 or greater, 14 by a fold-change of 50, and 2 by a fold-change of 100 in at least 1 pair, suggesting large differences in the proteins present in these fractions. Even though the classical MFGM proteins were enriched in the MFGM fractions, the relative protein composition varied considerably, and all contain an abundance of milk (casein and whey) proteins. Lipidomic analysis identified a total of 393 lipid species across both positive and negative ionization modes, with the major classes detected being triglycerides, sphingomyelins, and several phospholipids. Across all samples, triglycerides comprised at least 50% of total lipids, with phosphatidylcholine and sphingomyelin being the second and third most abundant lipid classes, respectively. These findings demonstrate the heterogeneous nature of various bovine commercial MFGM fractions. This variation must be considered when evaluating and describing potential functional benefits of these products shown in clinical trials.
Asunto(s)
Glucolípidos , Glicoproteínas , Leche/química , Animales , Caseínas/análisis , Bovinos , Cromatografía Liquida , Humanos , Lactante , Fórmulas Infantiles/química , Gotas Lipídicas , Membranas , Proteómica/métodos , Espectrometría de Masas en Tándem , Triglicéridos/análisis , Triglicéridos/química , Proteína de Suero de Leche/análisisRESUMEN
State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (µDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the µDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the µDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The µDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked µDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed µDIA provided 24% more true positives at the same false positive rate.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Péptidos/análisis , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida , Bases de Datos de Proteínas , Escherichia coli/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Programas Informáticos , Flujo de TrabajoRESUMEN
The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.
Asunto(s)
Arritmias Cardíacas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Potenciales de Acción , Secuencia de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Regulación de la Expresión Génica , Células HEK293 , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/patología , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Miocardio/metabolismo , Miocardio/patología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Transducción de SeñalRESUMEN
The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.
Asunto(s)
Síndrome de Brugada/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Canales de Sodio/metabolismo , Animales , Síndrome de Brugada/genética , Síndrome de Brugada/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Células HEK293 , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Activación del Canal Iónico/genética , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/patología , Potenciales de la Membrana/genética , Ratones , Proteínas Musculares/genética , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5 , Fosforilación/genética , Estructura Terciaria de Proteína , Canales de Sodio/genéticaRESUMEN
The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.
Asunto(s)
Arritmias Cardíacas/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Miocardio/enzimología , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Procesamiento Proteico-Postraduccional , Potenciales de Acción , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/terapia , Predisposición Genética a la Enfermedad , Humanos , Activación del Canal Iónico , Cinética , Mutación , Canal de Sodio Activado por Voltaje NAV1.5/genética , Fenotipo , Fosforilación , Pronóstico , Sodio/metabolismoRESUMEN
PURPOSE: Nonsyndromic craniosynostosis (NCS), the premature fusion of cranial sutures, results in an abnormal skull shape and is associated with a significant morbidity. Proteomics is a promising tool for disease characterization and biomarker discovery; we aimed to identify biologically relevant differentially expressed proteins for NCS. EXPERIMENTAL DESIGN: Label-based quantitative proteomic profiling using TMT was performed on protein extracted from mesenchymal stem cells, osteoblasts and bone tissue of five open and five fused sutures of sagittal NCS (sNCS) and analyzed using quantitative LC-MS/MS based bottom-up proteomics. Differential protein abundance between open and fused sutures was determined to identify biologically relevant proteins of interest. Proteins were validated in an independent sample set by western blot and immunohistochemistry. RESULTS: We observed 838 differentially expressed proteins between open and fused sutures of sNCS. Decorin, lumican, and asporin were significantly downregulated while COL4A1 and TGFß1|1 were upregulated in fused compared to open sutures. CONCLUSIONS AND CLINICAL RELEVANCE: The majority of significantly differentially expressed proteins between open and fused sutures were observed in the proteomes of osteoblasts suggesting that protein changes contributing to premature sagittal suture fusion occur predominantly at the osteoblast level. Our findings suggest a possible ineffective ECM deposition at the osteoblast cell stage.
Asunto(s)
ProteómicaRESUMEN
[This corrects the article DOI: 10.3389/fmolb.2020.600840.].
RESUMEN
Serum-based biomarkers hold propitious applications for addressing livestock health, and management. However, discovery of protein biomarkers in complex biological fluids like serum is wholly intractable due to the large dynamic range of protein concentrations; that is, Ë10-12 high abundance proteins constitute >90% of the total protein content and effectively mask proteomic detection of low-abundance biomarkers. Toward addressing this limitation, we test a continuous elution size-based fractionation method, and two approaches that use affinity interaction-based separation of proteins in preparing bovine serum, and compare liquid chromatography tandem mass spectrometry protein identification to neat serum. Our results identify the high-abundance proteins in bovine serum, and demonstrate dynamic range compression and improved protein identification with the different enrichment methods. Although these findings indicate the highest protein number identified in bovine serum (445 proteins, all methods combined), and by any single sample processing method (312 proteins) to date, they still remain lower than levels deemed necessary for biomarker discovery. As such, this investigation revealed limitations to resolving the bovine serum proteome, and the need for species-specific tools for immunodepleting high-abundance proteins. In concert, this study represents a step toward advancing sample preparation methods for bovine serum biomarker identification.
Asunto(s)
Proteínas Sanguíneas/análisis , Fraccionamiento Químico/métodos , Cromatografía Liquida/métodos , Proteoma/análisis , Suero/química , Espectrometría de Masas en Tándem/métodos , Animales , Biomarcadores/sangre , Bovinos , Femenino , Manejo de Especímenes/métodosRESUMEN
Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder associated with premutation CGG-repeat expansions (55-200 repeats) in the 5' non-coding portion of the fragile X mental retardation 1 (FMR1) gene. Core features of FXTAS include progressive tremor/ataxia, cognitive decline, variable brain volume loss, and white matter disease. The principal histopathological feature of FXTAS is the presence of central nervous system (CNS) and non-CNS intranuclear inclusions. Objective: To further elucidate the molecular underpinnings of FXTAS through the proteomic characterization of human FXTAS cortexes. Results: Proteomic analysis of FXTAS brain cortical tissue (n = 8) identified minor differences in protein abundance compared to control brains (n = 6). Significant differences in FXTAS relative to control brain predominantly involved decreased abundance of proteins, with the greatest decreases observed for tenascin-C (TNC), cluster of differentiation 38 (CD38), and phosphoserine aminotransferase 1 (PSAT1); proteins typically increased in other neurodegenerative diseases. Proteins with the greatest increased abundance include potentially novel neurodegeneration-related proteins and small ubiquitin-like modifier 1/2 (SUMO1/2). The FMRpolyG peptide, proposed in models of FXTAS pathogenesis but only identified in trace amounts in the earlier study of FXTAS inclusions, was not identified in any of the FXTAS or control brains in the current study. Discussion: The observed proteomic shifts, while generally relatively modest, do show a bias toward decreased protein abundance with FXTAS. Such shifts in protein abundance also suggest altered RNA binding as well as loss of cell-cell adhesion/structural integrity. Unlike other neurodegenerative diseases, the proteome of end-stage FXTAS does not suggest a strong inflammation-mediated degenerative response.
RESUMEN
Trophectoderm of blastocysts mediate early events in fetal-maternal communication, enabling implantation and establishment of a functional placenta. Inadequate or impaired developmental events linked to trophoblasts directly impact early embryo survival and successful implantation during a crucial period that corresponds with high incidence of pregnancy losses in dairy cows. As yet, the molecular basis of bovine trophectoderm development and signaling towards initiation of implantation remains poorly understood. In this study, we developed methods for culturing undifferentiated bovine blastocyst-derived trophoblasts and used both transcriptomics and proteomics in early colonies to categorize and elucidate their functional characteristics. A total of 9270 transcripts and 1418 proteins were identified and analyzed based on absolute abundance. We profiled an extensive list of growth factors, cytokines and other relevant factors that can effectively influence paracrine communication in the uterine microenvironment. Functional categorization and analysis revealed novel information on structural organization, extracellular matrix composition, cell junction and adhesion components, transcription networks, and metabolic preferences. Our data showcase the fundamental physiology of bovine trophectoderm and indicate hallmarks of the self-renewing undifferentiated state akin to trophoblast stem cells described in other species. Functional features uncovered are essential for understanding early events in bovine pregnancy towards initiation of implantation.
RESUMEN
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55-200 CGG repeats) in the 5' noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.
Asunto(s)
Ataxia/genética , Ataxia/patología , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/patología , Lóbulo Frontal/patología , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/patología , Temblor/genética , Temblor/patología , Secuencia de Aminoácidos , Ataxia/metabolismo , Femenino , Citometría de Flujo/métodos , Síndrome del Cromosoma X Frágil/metabolismo , Lóbulo Frontal/metabolismo , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Masculino , Proteómica/métodos , Temblor/metabolismoRESUMEN
Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.
Asunto(s)
Matriz Extracelular/metabolismo , Ventrículos Cardíacos/metabolismo , Nodo Sinoatrial/metabolismo , Animales , Membrana Basal/química , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Relojes Biológicos/fisiología , Fenómenos Biomecánicos , Colágeno/metabolismo , Colágeno/ultraestructura , Elasticidad , Elastina/metabolismo , Elastina/ultraestructura , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Fibronectinas/metabolismo , Fibronectinas/ultraestructura , Técnica del Anticuerpo Fluorescente , Ventrículos Cardíacos/química , Ventrículos Cardíacos/ultraestructura , Espectrometría de Masas , Microscopía de Fuerza Atómica , Microscopía Electroquímica de Rastreo , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Proteoma , Proteómica , Nodo Sinoatrial/química , Nodo Sinoatrial/ultraestructura , Porcinos , Resistencia a la TracciónRESUMEN
BACKGROUND: Atherosclerotic animal models show increased recruitment of inflammatory cells to the heart after myocardial infarction (MI), which impacts ventricular function and remodeling. OBJECTIVE: The purpose of this study was to determine whether increased myocardial inflammation after MI also contributes to arrhythmias. METHODS: MI was created in 3 mouse models: (1) atherosclerotic (apolipoprotein E deficient [ApoE(-/-)] on atherogenic diet, n = 12); (2) acute inflammation (wild-type [WT] given daily lipopolysaccharide [LPS] 10 µg/day, n = 7); and (3) WT (n = 14). Sham-operated (n = 4) mice also were studied. Four days post-MI, an inflammatory protease-activatable fluorescent probe (Prosense680) was injected intravenously to quantify myocardial inflammation on day 5. Optical mapping with voltage-sensitive dye was performed on day 5 to assess electrophysiology and arrhythmia susceptibility. RESULTS: Inflammatory activity (Prosense680 fluorescence) was increased approximately 2-fold in ApoE+MI and LPS+MI hearts vs WT+MI (P<.05) and 3-fold vs sham (P<.05). ApoE+MI and LPS+MI hearts also had prolonged action potential duration, slowed conduction velocity, and increased susceptibility to pacing-induced arrhythmias (56% and 71% vs 13% for WT+MI and 0% for sham, respectively, P<.05, for ApoE+MI and LPS+MI groups vs both WT+MI and sham). Increased macrophage accumulation in ApoE+MI and LPS+MI hearts was confirmed by immunofluorescence. Macrophages were associated with areas of connexin43 (Cx43) degradation, and a 2-fold decrease in Cx43 expression was found in ApoE+MI vs WT+MI hearts (P<.05). ApoE+MI hearts also had a 3-fold increase in interleukin-1ß expression, an inflammatory cytokine known to degrade Cx43. CONCLUSION: Underlying atherosclerosis exacerbates post-MI electrophysiological remodeling and arrhythmias. LPS+MI hearts fully recapitulate the atherosclerotic phenotype, suggesting myocardial inflammation as a key contributor to post-MI arrhythmia.