RESUMEN
Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.
Asunto(s)
Estructura Secundaria de Proteína/genética , Deficiencias en la Proteostasis/genética , Enfermedad de von Willebrand Tipo 2/genética , Factor de von Willebrand/genética , Sustitución de Aminoácidos , Plaquetas/química , Plaquetas/metabolismo , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Mutación con Pérdida de Función/genética , Espectrometría de Masas , Dominios Proteicos/genética , Pliegue de Proteína , Multimerización de Proteína/genética , Deficiencias en la Proteostasis/sangre , Deficiencias en la Proteostasis/patología , Enfermedad de von Willebrand Tipo 2/sangre , Enfermedad de von Willebrand Tipo 2/patología , Factor de von Willebrand/química , Factor de von Willebrand/ultraestructuraRESUMEN
Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, â¼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-ß and α-synuclein.
Asunto(s)
Amiloidosis/metabolismo , Catequina/análogos & derivados , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloide/biosíntesis , Catequina/farmacología , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Cadenas Ligeras de Inmunoglobulina/orina , Cinética , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.
Asunto(s)
Proteínas Portadoras/metabolismo , Ceramidas/metabolismo , Fosfatidilserinas/fisiología , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Línea Celular , Cristalografía por Rayos X , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Unión Proteica , Electricidad EstáticaRESUMEN
Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that â¼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Insulina/deficiencia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Fragmentos de Péptidos/metabolismo , Adulto , Animales , Autofagia , Proteínas Contráctiles/biosíntesis , Proteínas Contráctiles/genética , Diabetes Mellitus Experimental/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias Musculares/metabolismo , Peso Molecular , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
Growth and differentiation factor 11 (GDF11) is a transforming growth factor ß superfamily member with a controversial role in aging processes. We have developed a highly specific LC-MS/MS assay to quantify GDF11, resolved from its homolog, myostatin (MSTN), based on unique amino acid sequence features. Here, we demonstrate that MSTN, but not GDF11, declines in healthy men throughout aging. Neither GDF11 nor MSTN levels differ as a function of age in healthy women. In an independent cohort of older adults with severe aortic stenosis, we show that individuals with higher GDF11 were more likely to be frail and have diabetes or prior cardiac conditions. Following valve replacement surgery, higher GDF11 at surgical baseline was associated with rehospitalization and multiple adverse events. Cumulatively, our results show that GDF11 levels do not decline throughout aging but are associated with comorbidity, frailty, and greater operative risk in older adults with cardiovascular disease.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Enfermedades Cardiovasculares/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Miostatina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Biomarcadores/sangre , Proteínas Morfogenéticas Óseas/sangre , Proteínas Morfogenéticas Óseas/química , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/mortalidad , Cromatografía Liquida , Demografía , Femenino , Factores de Diferenciación de Crecimiento/sangre , Factores de Diferenciación de Crecimiento/química , Humanos , Masculino , Persona de Mediana Edad , Miostatina/sangre , Miostatina/química , Factores de Riesgo , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and ß-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of ß-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a ß-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteoma/metabolismo , Músculo Cuádriceps/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/biosíntesis , Aminoácidos/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/tratamiento farmacológico , Ácidos Grasos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Peróxido de Hidrógeno/metabolismo , Hipoglucemiantes/uso terapéutico , Immunoblotting , Insulina/uso terapéutico , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Consumo de Oxígeno , Proteómica , Transducción de SeñalRESUMEN
BACKGROUND: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions. METHODS: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment. RESULTS: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%). CONCLUSIONS: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/sangre , Mieloma Múltiple/sangre , Mieloma Múltiple/diagnóstico , Neoplasia Residual/sangre , Neoplasia Residual/diagnóstico , Péptidos/sangre , Médula Ósea/patología , Examen de la Médula Ósea , Regiones Determinantes de Complementariedad/sangre , Regiones Determinantes de Complementariedad/genética , Humanos , Cadenas Ligeras de Inmunoglobulina/genética , Mieloma Múltiple/patología , Neoplasia Residual/patología , Péptidos/genética , ARN Mensajero/sangre , ARN Mensajero/genética , SucciónRESUMEN
BACKGROUND: Myostatin is a protein synthesized and secreted by skeletal muscle that negatively regulates muscle mass. The extent to which circulating myostatin levels change in the context of aging is controversial, largely due to methodological barriers. METHODS: We developed a specific and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure concentrations of myostatin and two of its key inhibitors, follistatin-related gene (FLRG) protein and growth and serum protein-1 (GASP-1) in 80 younger (<40 years), 80 older (>65 years), and 80 sarcopenic older women and men. RESULTS: Older women had 34 % higher circulating concentrations of myostatin than younger women. Per unit of lean mass, both older and sarcopenic older women had >23 % higher myostatin levels than younger women. By contrast, younger men had higher myostatin concentrations than older men with and without sarcopenia. Younger men had approximately twofold higher concentrations of myostatin than younger women; however, older women and sarcopenic older women had significantly higher relative myostatin levels than the corresponding groups of men. In both sexes, sarcopenic older subjects had the highest concentrations of FLRG. Circulating concentrations of myostatin exhibited positive, but not robust, correlations with relative muscle mass in both sexes. CONCLUSIONS: Our data suggest that myostatin may contribute to the higher prevalence of sarcopenia in women but acts as a homeostatic regulator of muscle mass in men. Moreover, this new LC-MS/MS-based approach offers a means to determine the extent to which myostatin serves as a biomarker of muscle health in diverse conditions of muscle loss and deterioration.
RESUMEN
OBJECTIVE: Visceral white adipose tissue (WAT) expansion and macrophage accumulation are associated with metabolic dysfunction. Visceral WAT typically shows greater macrophage infiltration. Preadipocytes show varying proinflammatory expression profiles among WAT depots. The objective was to examine the secretomes and chemoattractive properties of preadipocytes from visceral and subcutaneous WAT. METHODS: A label-free quantitative proteomics approach was applied to study secretomes of subcutaneous and omental preadipocytes from obese subjects. Enzyme-linked immunosorbent assays and chemotaxis assays were used to confirm proinflammatory chemokine secretion between depots. RESULTS: Preadipocyte secretomes showed greater variation between depots than did intracellular protein expression. Chemokines were the most differentially secreted proteins. Omental preadipocytes induced chemoattraction of macrophages and monocytes. Neutralizing antibodies to the identified chemokines reduced macrophage/monocyte chemoattraction. Subcutaneous preadipocytes treated with interleukin-6 (IL-6) resembled omental preadipocytes in terms of chemokine secretion and macrophage/monocyte chemoattraction. Janus-activated kinase (JAK1/2) protein expression, which transduces IL-6 signaling, was higher in omental than subcutaneous preadipocytes and WAT. Inhibiting JAK in omental preadipocytes decreased chemokine secretion and macrophage/monocyte chemoattraction to levels closer to that observed in subcutaneous preadipocytes. CONCLUSIONS: Secretomes of omental and subcutaneous preadipocytes are distinct, with the former inducing more macrophage/monocyte chemoattraction, in part through IL-6/JAK-mediated signaling.
Asunto(s)
Adipocitos Blancos/metabolismo , Inflamación/metabolismo , Obesidad/metabolismo , Epiplón/metabolismo , Grasa Subcutánea/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-6/metabolismo , Grasa Intraabdominal/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Proteínas/metabolismoRESUMEN
Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise.
Asunto(s)
Ejercicio Físico/fisiología , Mitocondrias Musculares/fisiología , Proteínas Mitocondriales/fisiología , Músculo Esquelético/fisiología , Estrés Oxidativo/fisiología , Resistencia Física/fisiología , Acetilación , Adolescente , Adulto , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Proteolisis , Conducta Sedentaria , Adulto JovenRESUMEN
Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin ß1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2',3'-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer.
Asunto(s)
Antineoplásicos/farmacología , Azadirachta/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Neoplasias de la Próstata/patología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Adhesiones Focales/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Extractos Vegetales/farmacocinética , Extractos Vegetales/toxicidad , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.
Asunto(s)
Exosomas/química , Membrana Basal Glomerular/química , Enfermedades Renales/orina , Podocitos/química , Proteinuria/orina , Proteómica/métodos , Urinálisis , Orina/química , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Biomarcadores/orina , Estudios de Casos y Controles , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Enfermedades Renales/diagnóstico , Masculino , Datos de Secuencia Molecular , Proteinuria/diagnóstico , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Anoctamin 5 (ANO5) is a putative intracellular calcium-activated chloride channel. Recessive mutations in ANO5 cause primary skeletal muscle disorders (limb-girdle muscular dystrophy 2L and distal muscular dystrophy), which are phenotypically similar to dysferlinopathy, a muscular dystrophy due to dysferlin-encoding gene (DYSF) mutations. METHODS: This study reports the phenotype and genotype of seven unrelated patients with ANO5-muscular dystrophy. RESULTS: Three patients had amyloid deposition in muscle and two had cardiac involvement. An additional patient without skeletal muscle amyloidosis had cardiac involvement with septal hypokinesis and supraventricular tachycardia requiring ablation. Amyloid subtyping using laser capture microdissection and mass spectrometry-based proteomic analysis did not identify ANO5 or any fragment of ANO5 in the amyloid deposits, but detected other known amyloidogenic proteins. Three patients had myotonic discharges without clinical myotonia. Four ANO5 mutations are novel, including a heterozygous 0.4 Mb deletion involving the entire ANO5 gene. CONCLUSIONS: The results of the present study suggest that ANO5 mutations can be associated with amyloid deposition in muscle, but the nature of the amyloid deposits remains indeterminate, as does their relationship with cardiac involvement. ANO5 analysis should be considered in cases of muscle amyloid deposition of indeterminate etiology. Electrical myotonia can accompany ANO5-muscular dystrophy.
Asunto(s)
Canales de Cloruro/genética , Distrofias Musculares/genética , Distrofias Musculares/patología , Adulto , Anciano , Amiloidosis/genética , Amiloidosis/patología , Anoctaminas , Femenino , Genotipo , Humanos , Captura por Microdisección con Láser , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Mutación , Miocardio/patología , FenotipoRESUMEN
The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.
Asunto(s)
Apoptosis , Hexoquinasa/metabolismo , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas Represoras/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Ácido Edético/metabolismo , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Glucólisis , Hexoquinasa/genética , Proteínas de Interacción con los Canales Kv/genética , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Represoras/genética , TransfecciónRESUMEN
Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis.
Asunto(s)
Restricción Calórica , Mitocondrias/metabolismo , Recambio Mitocondrial/fisiología , Envejecimiento , Animales , ADN Mitocondrial/metabolismo , Regulación hacia Abajo , Complejo I de Transporte de Electrón/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Perfilación de la Expresión Génica , Ratones , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Estrés Oxidativo , Proteómica , TranscriptomaRESUMEN
Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of â¼3 µM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas de Interacción con los Canales Kv/metabolismo , Multimerización de Proteína/fisiología , Proteínas Represoras/metabolismo , Calcineurina/química , Calcineurina/genética , Calcineurina/metabolismo , Calcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Proteínas de Interacción con los Canales Kv/química , Proteínas de Interacción con los Canales Kv/genética , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: Self-harm is a common reason for Emergency Department (ED) attendance. We aimed to develop a clinical tool to help identify patients at higher risk of repeat self-harm, or suicide, within 6 months of an ED self-harm presentation. METHOD: The tool, the ReACT Self-Harm Rule, was derived using multicentre data from a prospective cohort study. Binary recursive partitioning was applied to data from two centres, and data from a separate centre were used to test the tool. There were 29 571 self-harm presentations to five hospital EDs between January 2003 and June 2007, involving 18 680 adults aged ⩾16 years. We estimated sensitivity, specificity and positive and negative predictive values to measure the performance of the tool. RESULTS: A self-harm presentation was classified as higher risk if at least one of the following factors was present: recent self-harm (in the past year), living alone or homelessness, cutting as a method of harm and treatment for a current psychiatric disorder. The rule performed with 95% sensitivity [95% confidence interval (CI) 94-95] and 21% specificity (95% CI 21-22), and had a positive predictive value of 30% (95% CI 30-31) and a negative predictive value of 91% (95% CI 90-92) in the derivation centres; it identified 83/92 of all subsequent suicides. CONCLUSIONS: The ReACT Self-Harm Rule might be used as a screening tool to inform the process of assessing self-harm presentations to ED. The four risk factors could also be used as an adjunct to in-depth psychosocial assessment to help guide risk formulation. The use of multicentre data helped to maximize the generalizability of the tool, but we need to further verify its external validity in other localities.
Asunto(s)
Escalas de Valoración Psiquiátrica/normas , Psicometría/instrumentación , Conducta Autodestructiva/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Servicio de Urgencia en Hospital/estadística & datos numéricos , Inglaterra/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Medición de Riesgo , Conducta Autodestructiva/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation. METHODS: We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides. RESULTS: We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls. CONCLUSIONS: Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1, asporin, cartilage oligomeric matrix protein, chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V, and proprotein convertase subtilisin/kexin 6.
Asunto(s)
Adenocarcinoma/sangre , Adenocarcinoma/genética , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/genética , ARN Mensajero/genética , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Masculino , Neoplasias de la Próstata/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Mortality, including suicide and accidents, is elevated in self-harm populations. Although risk factors for suicide following self-harm are often investigated, rarely have those for accidents been studied. Our aim was to compare risk factors for suicide and accidents. METHOD: A prospective cohort (n=30 202) from the Multicentre Study of Self-harm in England, 2000-2007, was followed up to 2010 using national death registers. Risk factors for suicide (intentional self-harm and undetermined intent) and accidents (narcotic poisoning, non-narcotic poisoning, and non-poisoning) following the last hospital presentation for self-harm were estimated using Cox models. RESULTS: During follow-up, 1833 individuals died, 378 (20.6%) by suicide and 242 (13.2%) by accidents. Independent predictors of both suicide and accidents were: male gender, age 35 years (except accidental narcotic poisoning) and psychiatric treatment (except accidental narcotic poisoning). Factors differentiating suicide from accident risk were previous self-harm, last method of self-harm (twofold increased risks for cutting and violent self-injury versus self-poisoning) and mental health problems. A risk factor specific to accidental narcotic poisoning was recreational/illicit drug problems, and a risk factor specific to accidental non-narcotic poisoning and non-poisoning accidents was alcohol involvement with self-harm. CONCLUSIONS: The similarity of risk factors for suicide and accidents indicates common experiences of socio-economic disadvantage, life problems and psychopathology resulting in a variety of self-destructive behaviour. Of factors associated with the accidental death groups, those for non-narcotic poisoning and other accidents were most similar to suicide; differences seemed to be related to criteria coroners use in reaching verdicts. Our findings support the idea of a continuum of premature death.
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Accidentes/mortalidad , Sistema de Registros , Conducta Autodestructiva/mortalidad , Trastornos Relacionados con Sustancias/mortalidad , Suicidio/estadística & datos numéricos , Adolescente , Adulto , Inglaterra/epidemiología , Femenino , Humanos , Masculino , Narcóticos/envenenamiento , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Factores de Riesgo , Conducta Autodestructiva/clasificación , Factores Socioeconómicos , Factores de Tiempo , Adulto JovenRESUMEN
The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.