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BACKGROUND: Glucose overload drives diabetic cardiomyopathy by affecting the tricarboxylic acid pathway. However, it is still unknown how cells could overcome massive chronic glucose influx on cellular and structural level. METHODS/MATERIALS: Expression profiles of hyperglycemic, glucose transporter-4 (GLUT4) overexpressing H9C2 (KE2) cardiomyoblasts loaded with 30 mM glucose (KE230L) and wild type (WT) cardiomyoblasts loaded with 30 mM glucose (WT30L) were compared using proteomics, real-time polymerase quantitative chain reaction analysis, or Western blotting, and immunocytochemistry. RESULTS: The findings suggest that hyperglycemic insulin-sensitive cells at the onset of diabetic cardiomyopathy present complex changes in levels of structural cell-related proteins like tissue inhibitor of metalloproteases-1 (1.3 fold), intercellular adhesion molecule 1 (1.8 fold), type-IV-collagen (3.2 fold), chaperones (Glucose-Regulated Protein 78: 1.8 fold), autophagy (Autophagosome Proteins LC3A, LC3B: 1.3 fold), and in unfolded protein response (UPR; activating transcription factor 6α expression: 2.3 fold and processing: 2.4 fold). Increased f-actin levels were detectable with glucose overload by immnocytochemistry. Effects on energy balance (1.6 fold), sirtuin expression profile (Sirtuin 1: 0.7 fold, sirtuin 3: 1.9 fold, and sirtuin 6: 4.2 fold), and antioxidant enzymes (Catalase: 0.8 fold and Superoxide dismutase 2: 1.5 fold) were detected. CONCLUSION: In conclusion, these findings implicate induction of chronic cell distress by sustained glucose accumulation with a non-compensatory repair reaction not preventing final cell death. This might explain the chronic long lasting pathogenesis observed in developing heart failure in diabetes mellitus.
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Cardiomiopatias Diabéticas , Transportador de Glucose Tipo 4 , Glucose , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 4/genética , Glucose/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Animais , Ratos , Linhagem Celular , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Hiperglicemia/metabolismo , AutofagiaRESUMO
Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.
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Desmina , Fibras Musculares Esqueléticas , Animais , Desmina/metabolismo , Desmina/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Introdução de Genes , Prótons , Mitocôndrias/metabolismo , Distrofias Musculares , CardiomiopatiasRESUMO
AIMS: Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins. METHODS: Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification. The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation. RESULTS: More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones. Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA). CONCLUSIONS: In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillogenesis is tightly regulated by the BAG3-associated protein quality system.
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Doenças do Sistema Nervoso Periférico , Humanos , Doenças do Sistema Nervoso Periférico/metabolismo , Actinas/análise , Actinas/metabolismo , Proteômica , Proteínas Musculares/metabolismo , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson's disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SNSurr.) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.
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Doença por Corpos de Lewy , Proteoma , Humanos , alfa-Sinucleína , ProteômicaRESUMO
Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.
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Cardiomiopatias , Desmina , Hexoquinase , Aminoácidos/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Citratos/metabolismo , Creatina Quinase Mitocondrial/metabolismo , Desmina/genética , Desmina/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Fosforilação Oxidativa , ProteômicaRESUMO
Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals.
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Proteômica , Tirosina 3-Mono-Oxigenase , Humanos , Lisossomos/metabolismo , Melaninas/metabolismo , Proteômica/métodos , Grânulos de Estresse , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.
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Cardiomiopatias Diabéticas , Hiperglicemia , Animais , Ciclo do Ácido Cítrico , Cardiomiopatias Diabéticas/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Lactatos/metabolismo , Miócitos Cardíacos/metabolismo , Necrose/metabolismo , RatosRESUMO
AIMS: We investigated N471D WASH complex subunit strumpellin (Washc5) knock-in and Washc5 knock-out mice as models for hereditary spastic paraplegia type 8 (SPG8). METHODS: We generated heterozygous and homozygous N471D Washc5 knock-in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. RESULTS: Homozygous N471D Washc5 knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASHC5-related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock-out mice showing normal WASHC5 levels could not be bred to homozygosity. CONCLUSIONS: While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.
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Proteômica , Paraplegia Espástica Hereditária , Animais , Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Mutação , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications.
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OBJECTIVE: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM). METHODS: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis. RESULTS: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc. CONCLUSIONS: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.
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Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.
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For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts >5µg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector® all-in-one solution with the Smart Protein Layers (SPL) approach.
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Western Blotting , Proteínas/análise , Proteoma , Proteômica , Animais , Western Blotting/normas , Calibragem , Humanos , Proteômica/normas , Padrões de Referência , Projetos de Pesquisa , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Data-independent acquisition (DIA) has recently developed as a powerful tool to enhance the quantification of peptides and proteins within a variety of sample types, by overcoming the stochastic nature of classical data-dependent approaches, as well as by enabling the identification of all peptides detected in a mass spectrometric event. Here, we describe a workflow for the establishment of a sample-fitting DIA method using Spectronaut Pulsar X (Biognosys, Switzerland).
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Proteínas/análise , Proteoma , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Projetos de Pesquisa , Fluxo de TrabalhoRESUMO
Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson's disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.
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Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Humanos , Microdissecção e Captura a Laser , Lasers , Melaninas , Proteômica/métodos , Substância Negra/metabolismoRESUMO
The pathological hallmark of Parkinson's disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes.
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Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Proteoma/metabolismo , Substância Negra/metabolismo , Sinaptossomos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Masculino , Melaninas/metabolismo , Metaloendopeptidases/metabolismo , Metionina tRNA Ligase/metabolismo , Proteômica/métodos , Timidina Quinase/metabolismoRESUMO
A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.
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Células-Tronco Pluripotentes Induzidas/virologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/fisiologia , Linhagem Celular , Doxiciclina/farmacologia , Humanos , Modelos Biológicos , Miócitos Cardíacos/virologia , Ativação Viral/efeitos dos fármacosRESUMO
Filamin C (FLNc) is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familial and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from FLNc and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: in comparison to wildtype animals (i) the expression level of FLNc protein is drastically reduced; (ii) mutant FLNc is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack Bcl2-associated athanogene 3 (BAG3) protein; (iv) the expression of heat shock protein beta-7 (HSPB7) is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective FLNc dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative, more amino-terminally located dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that FLNc is important for mechanosensing in the context of Z-disc stabilization and maintenance.
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Filaminas/genética , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Sarcômeros/patologia , Animais , Técnicas de Introdução de Genes , Homozigoto , Camundongos , Mutação , Miopatias Congênitas Estruturais/metabolismo , Sarcômeros/metabolismoRESUMO
Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue.
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Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteoma/metabolismo , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Miosite de Corpos de Inclusão/patologia , Agregados Proteicos/fisiologia , Estabilidade Proteica , Proteoma/análise , Proteoma/genéticaRESUMO
Evidence has shown that protein supplementation following resistance exercise training (RET) helps to further enhance muscle mass and strength. Studies have demonstrated that collagen peptides containing mostly non-essential amino acids increase fat-free mass (FFM) and strength in sarcopenic men. The aim of this study was to investigate whether collagen peptide supplementation in combination with RET influences the protein composition of skeletal muscle. Twenty-five young men (age: 24.2 ± 2.6 years, body mass (BM): 79.6 ± 5.6 kg, height: 185.0 ± 5.0 cm, fat mass (FM): 11.5% ± 3.4%) completed body composition and strength measurements and vastus lateralis biopsies were taken before and after a 12-week training intervention. In a double-blind, randomized design, subjects consumed either 15 g of specific collagen peptides (COL) or a non-caloric placebo (PLA) every day within 60 min after their training session. A full-body hypertrophy workout was completed three times per week and included four exercises using barbells. Muscle proteome analysis was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). BM and FFM increased significantly in COL compared with PLA, whereas no differences in FM were detected between the two groups. Both groups improved in strength levels, with a slightly higher increase in COL compared with PLA. In COL, 221 higher abundant proteins were identified. In contrast, only 44 proteins were of higher abundance in PLA. In contrast to PLA, the upregulated proteins in COL were mostly associated with the protein metabolism of the contractile fibers. In conclusion, the use of RET in combination with collagen peptide supplementation results in a more pronounced increase in BM, FFM, and muscle strength than RET alone. More proteins were upregulated in the COL intervention most of which were associated with contractile fibers.
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Colágeno/administração & dosagem , Suplementos Nutricionais , Força Muscular/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteoma , Treinamento Resistido , Adulto , Composição Corporal/efeitos dos fármacos , Método Duplo-Cego , Exercício Físico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Fenômenos Fisiológicos da Nutrição Esportiva , Adulto JovemRESUMO
In a common proteomics analysis today, the origins of our sample in the vial are known and therefore a database dependent approach to identify the containing peptides can be used. The first YPIC challenge though provided us with 19 synthetic peptides, which together formed an English sentence. For the identification of these peptides, a de-novo approach was used, which brought us together with an internet search engine to the hidden sentence. But only having the sentence was not sufficient for us, we also wanted to identify as many as possible of the spectra in our data. Therefore, we created and refined a database approach from the de-novo method and finally could identify the peptide-sentence with a good overlap.