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While Aß and Tau cellular distribution has been largely studied, the comparative internalization and subcellular accumulation of Tau and Aß isolated from human brain extracts in endothelial and neuronal cells has not yet been unveiled. We have previously demonstrated that controlled enrichment of Aß from human brain extracts constitutes a valuable tool to monitor cellular internalization in vitro and in vivo. Herein, we establish an alternative method to strongly enrich Aß and Tau aggregates from human AD brains, which has allowed us to study and compare the cellular internalization, distribution and toxicity of both proteins within brain barrier endothelial (bEnd.3) and neuronal (Neuro2A) cells. Our findings demonstrate the suitability of human enriched brain extracts to monitor the intracellular distribution of human Aß and Tau, which, once internalized, show dissimilar sorting to different organelles within the cell and differential toxicity, exhibiting higher toxic effects on neuronal cells than on endothelial cells. While tau is strongly concentrated preferentially in mitochondria, Aß is distributed predominantly within the endolysosomal system in endothelial cells, whereas the endoplasmic reticulum was its preferential location in neurons. Altogether, our findings display a picture of the interactions that human Aß and Tau might establish in these cells.
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Péptidos beta-Amiloides , Células Endoteliales , Neuronas , Proteínas tau , Humanos , Proteínas tau/metabolismo , Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Células Endoteliales/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Animales , Ratones , Mitocondrias/metabolismo , Línea CelularRESUMEN
BACKGROUND: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. METHODS: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. RESULTS: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. CONCLUSIONS: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.
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As neurons age, protein homeostasis becomes less efficient, resulting in misfolding and aggregation. Chaperone proteins perform vital functions in the maintenance of cellular proteostasis, and chaperone-based therapies that promote sequestration of toxic aggregates may prove useful in blocking the development of neurodegenerative disease. We previously demonstrated that proSAAS, a small secreted neuronal protein, exhibits potent chaperone activity against protein aggregation in vitro and blocks the cytotoxic effects of amyloid and synuclein oligomers in cell culture systems. We now examine whether cytoplasmic expression of proSAAS results in interactions with protein aggregates in this cellular compartment. We report that expression of proSAAS within the cytoplasm generates dense, membraneless 2 µm proSAAS spheres which progressively fuse to form larger spheres, suggesting liquid droplet-like properties. ProSAAS spheres selectively accumulate a C-terminally truncated fluorescently tagged form of TDP-43, initiating its cellular redistribution; these TDP-43-containing spheres also exhibit dynamic fusion. Efficient encapsulation of TDP-43 into proSAAS spheres is driven by its C-terminal prion-like domain; spheres must be formed for sequestration to occur. Three proSAAS sequences, a predicted coiled-coil, a conserved region (residues 158-169), and the positively charged sequence 181-185, are all required for proSAAS to form spheres able to encapsulate TDP-43 aggregates. Substitution of lysines for arginines in the 181-185 sequence results in nuclear translocation of proSAAS and encapsulation of nuclear-localized TDP-43216-414. As a functional output, we demonstrate that proSAAS expression results in cytoprotection against full-length TDP-43 toxicity in yeast. We conclude that proSAAS can act as a functional holdase for TDP-43 via this phase-separation property, representing a cytoprotectant whose unusual biochemical properties can potentially be exploited in the design of therapeutic molecules.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Esclerosis Amiotrófica Lateral/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Chaperonas Moleculares/genética , Agregado de ProteínasRESUMEN
The fact that cerebrospinal fluid (CSF) deeply irrigates the brain together with the relative simplicity of sample extraction from patients make this biological fluid the best target for biomarker discovery in neurodegenerative diseases. During the last decade, biomarker discovery has been especially fruitful for the identification new proteins that appear in the CSF of Alzheimer's disease (AD) patients together with amyloid-ß (Aß42), total tau (T-tau), and phosphorylated tau (P-tau). Thus, several proteins have been already stablished as important biomarkers, due to an increase (i.e., CHI3L1) or a decrease (i.e., VGF) in AD patients' CSF. Notwithstanding this, only a deep analysis of a database generated with all the changes observed in CSF across multiple proteomic studies, and especially those using state-of-the-art methodologies, may expose those components or metabolic pathways disrupted at different levels in AD. Deep comparative analysis of all the up- and down-regulated proteins across these studies revealed that 66% of the most consistent protein changes in CSF correspond to intracellular proteins. Interestingly, processes such as those associated to glucose metabolism or RXR signaling appeared inversely represented in CSF from AD patients in a significant manner. Herein, we discuss whether certain cellular processes constitute accurate indicators of AD progression by examining CSF. Furthermore, we uncover new CSF AD markers, such as ITAM, PTPRZ or CXL16, identified by this study.
RESUMEN
BACKGROUND: During the last two decades, over 100 proteomics studies have identified a variety of potential biomarkers in CSF of Alzheimer's (AD) patients. Although several reviews have proposed specific biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specific up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteomics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identified in each study are consistent in distribution; direction change; and significance, which would strengthen their potential use in studies of AD pathology and progression. METHODS: We generated a database containing all CSF proteins whose levels are known to be significantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. RESULTS: Of the 162 proteins that were identified in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identified 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fingerprinting revealed specific peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. CONCLUSIONS: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical significance; this panel constitutes a potent tool for the classification and diagnosis of AD.
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BACKGROUND: Intracerebral inoculation of extracts from post-mortem human Alzheimer's disease brains into mice produces a prion-like spreading effect of amyloid-ß. The differences observed between these extracts and the synthetic peptide, in terms of amyloid-ß internalization and seed and cell-to-cell transmission of cytosolic protein aggregates, suggest that brain extracts contain key contributors that enhance the prion-like effect of amyloid-ß. Nevertheless, these potential partners are still unknown due to the complexity of whole brain extracts. METHODS: Herein, we established a method based on sequential detergent solubilization of post-mortem samples of human brains affected by Alzheimer's disease that strongly enrich amyloid-ß aggregates by eliminating 92% of the remaining proteins. Internalization of Aß1-42 from the enriched AD extracts was evaluated in vitro, and internalization of fluorescent-labeled AD extracts was also investigated in vivo. Furthermore, we carried out a molecular characterization of the Aß-enriched fraction using label-free proteomics, studying the distribution of representative components in the amygdala and the olfactory cortex of additional human AD brain samples by immunohistochemistry. RESULTS: Aß1-42 from the enriched AD extracts are internalized into endothelial cells in vitro after 48 h. Furthermore, accumulation of fluorescent-labeled Aß-enriched extracts into mouse microglia was observed in vivo after 4 months of intracerebral inoculation. Label-free proteomics (FDR < 0.01) characterization of the amyloid-ß-enriched fraction from different post-mortem samples allowed for the identification of more than 130 proteins, several of which were significantly overrepresented (i.e., ANXA5 and HIST1H2BK; p < 0.05) and underrepresented (i.e., COL6A or FN1; p < 0.05) in the samples with Alzheimer's disease. We were also able to identify proteins exclusively observed in Alzheimer's disease (i.e., RNF213) or only detected in samples not affected by the disease (i.e., CNTN1) after the enrichment process. Immunohistochemistry against these proteins in additional tissues revealed their particular distribution in the amygdala and the olfactory cortex in relation to the amyloid-ß plaque. CONCLUSIONS: Identification and characterization of the unique features of these extracts, in terms of amyloid-ß enrichment, identification of the components, in vitro and in vivo cell internalization, and tissue distribution, constitute the best initial tool to further investigate the seeding and transmissibility proposed in the prion-like hypothesis of Alzheimer's disease.
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Enfermedad de Alzheimer/metabolismo , Amígdala del Cerebelo/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Corteza Olfatoria/metabolismo , Fragmentos de Péptidos/metabolismo , Proteómica , Bancos de Tejidos , Enfermedad de Alzheimer/patología , Amígdala del Cerebelo/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales , Femenino , Hipocampo/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Microvasos , Corteza Olfatoria/patología , Priones/metabolismoRESUMEN
Structural and functional abnormalities in the cerebral microvasculature have been observed in Alzheimer's disease (AD) patients and animal models. One cause of hypoperfusion is the thickening of the cerebrovascular basement membrane (CVBM) due to increased collagen-IV deposition around capillaries. This study investigated whether these and other alterations in the cerebrovascular system associated with AD can be prevented by long-term dietary supplementation with the antioxidant ubiquinol (Ub) stabilized with Kaneka QH P30 powder containing ascorbic acid (ASC) in a mouse model of advanced AD (3â¯×â¯Tg-AD mice, 12â¯months old). Animals were treated from prodromal stages of disease (3â¯months of age) with standard chow without or with Ubâ¯+â¯ASC or ASC-containing vehicle and compared to wild-type (WT) mice. The number of ß-amyloid (Aß) plaques in the hippocampus and entorhinal cortex was higher in female than in male 3â¯×â¯Tg-AD mice. Extensive regions of hypoxia were characterized by a higher plaque burden in females only. This was abolished by Ubâ¯+â¯ASC and, to a lesser extent, by ASC treatment. Irrespective of Aß burden, increased collagen-IV deposition in the CVBM was observed in both male and female 3â¯×â¯Tg-AD mice relative to WT animals; this was also abrogated in Ubâ¯+â¯ASC- and ASC-treated mice. The chronic inflammation in the hippocampus and oxidative stress in peripheral leukocytes of 3â¯×â¯Tg-AD mice were likewise reversed by antioxidant treatment. These results provide strong evidence that long-term antioxidant treatment can mitigate plasma oxidative stress, amyloid burden, and hypoxia in the AD brain parenchyma.
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Enfermedad de Alzheimer/tratamiento farmacológico , Antioxidantes/uso terapéutico , Ácido Ascórbico/uso terapéutico , Placa Amiloide/tratamiento farmacológico , Ubiquinona/análogos & derivados , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Hipoxia de la Célula , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/metabolismo , Corteza Entorrinal/patología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ubiquinona/farmacología , Ubiquinona/uso terapéuticoRESUMEN
Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.
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Actinas/genética , Miosina Tipo I/genética , Vesículas Secretoras/metabolismo , Actinas/metabolismo , Animales , Transporte Biológico , Células COS , Proteínas Portadoras , Chlorocebus aethiops , Aparato de Golgi/metabolismo , Ratones , Miosina Tipo I/metabolismo , Células Neuroendocrinas/metabolismo , Sistemas Neurosecretores/metabolismo , Células PC12 , Unión Proteica , RatasRESUMEN
During their lifetime, females are subjected to different nutritional and hormonal factors that could increase the risk of obesity and associated comorbidities. From early postnatal periods until the postmenopausal phase, exposure to over nutrition, high-energy diet and oestrogen deficiency, are considered as significant obesity risk factors in women. In this study, we assessed how key transitional life events and exposure to different nutrition influence energy homeostasis in a rat model. Specifically, we assessed the sequential exposure to postnatal over nutrition, high-fat diet (HFD) after weaning, followed later by ovariectomy (OVX; as a model of menopause). Each obesity risk factor increased significantly body weight (BW) and adiposity, with additive effects after sequential exposure. Increased energy intake in both HFD and/or OVX groups, and decreased locomotor activity and energy expenditure after OVX can explain these metabolic changes. Our study also documents decreased lipogenic pathway in mesenteric adipose tissue after HFD and/or OVX, independent of previous postnatal programming, yet only HFD evoked this effect in liver. In addition, we report an increase in the expression of the hepatic PEPCK depending on previous metabolic status. Overall, our results identify the impact of different risk factors, which will help in understanding the development of obesity in females.
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Tejido Adiposo/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Animales , Composición Corporal , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ingestión de Energía , Metabolismo Energético , Femenino , Locomoción , Mesenterio , Ovariectomía , Fenotipo , Ratas , Factores de Riesgo , Factores SexualesRESUMEN
Adipose-tissue (AT) is an endocrine organ that dynamically secretes multiple hormones, the adipokines, which regulate key physiological processes. However, adipokines and their receptors are also expressed and regulated in other tissues, including the pituitary, suggesting that locally- and AT-produced adipokines might comprise a regulatory circuit that relevantly modulate pituitary cell-function. Here, we used primary pituitary cell-cultures from two normal nonhuman-primate species [Papio-anubis/Macaca-fascicularis] to determine the impact of different adipokines on the functioning of all anterior-pituitary cell-types. Leptin and resistin stimulated GH-release, a response that was blocked by somatostatin. Conversely, adiponectin decreased GH-release, and inhibited GHRH-, but not ghrelin-stimulated GH-secretion. Furthermore: 1) Leptin stimulated PRL/ACTH/FSH- but not LH/TSH-release; 2) adiponectin stimulated PRL-, inhibited ACTH- and did not alter LH/FSH/TSH-release; and 3) resistin increased ACTH-release and did not alter PRL/LH/FSH/TSH-secretion. These effects were mediated through the activation of common (AC/PKA) and distinct (PLC/PKC, intra-/extra-cellular calcium, PI3K/MAPK/mTOR) signaling-pathways, and by the gene-expression regulation of key receptors/transcriptional-factors involved in the functioning of these pituitary cell-types (e.g. GHRH/ghrelin/somatostatin/insulin/IGF-I-receptors/Pit-1). Finally, we found that primate pituitaries expressed leptin/adiponectin/resistin. Altogether, these and previous data suggest that local-production of adipokines/receptors, in conjunction with circulating adipokine-levels, might comprise a relevant regulatory circuit that contribute to the fine-regulation of pituitary functions.
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Adiponectina/metabolismo , Adenohipófisis/citología , Adenohipófisis/metabolismo , Hormonas Hipofisarias/biosíntesis , Adipoquinas/metabolismo , Adipoquinas/farmacología , Adiponectina/farmacología , Animales , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Leptina/metabolismo , Leptina/farmacología , Papio , Adenohipófisis/efectos de los fármacos , Primates , Resistina/metabolismo , Resistina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Adipose tissue (AT) dysfunction in obesity is commonly linked to insulin resistance and promotes the development of metabolic disease. Bariatric surgery (BS) represents an effective strategy to reduce weight and to improve metabolic health in morbidly obese subjects. However, the mechanisms and pathways that are modified in AT in response to BS are not fully understood, and few information is still available as to whether these may vary depending on the metabolic status of obese subjects. METHODS: Abdominal subcutaneous adipose tissue (SAT) samples were obtained from morbidly obese women (n = 18) before and 13.3 ± 0.37 months after BS. Obese women were stratified into two groups: normoglycemic (NG; Glu < 100 mg/dl, HbA1c <5.7 %) or insulin resistant (IR; Glu 100-126 mg/dl, HbA1c 5.7-6.4 %) (n = 9/group). A multi-comparative proteomic analysis was employed to identify differentially regulated SAT proteins by BS and/or the degree of insulin sensitivity. Serum levels of metabolic, inflammatory, and anti-oxidant markers were also analyzed. RESULTS: Before surgery, NG and IR subjects exhibited differences in AT proteins related to inflammation, metabolic processes, the cytoskeleton, and mitochondria. BS caused comparable weight reductions and improved glucose homeostasis in both groups. However, BS caused dissimilar changes in metabolic enzymes, inflammatory markers, cytoskeletal components, mitochondrial proteins, and angiogenesis regulators in NG and IR women. CONCLUSIONS: BS evokes significant molecular rearrangements indicative of improved AT function in morbidly obese women at either low or high metabolic risk, though selective adaptive changes in key cellular processes occur depending on the initial individual's metabolic status.
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Biomarcadores/metabolismo , Resistencia a la Insulina , Síndrome Metabólico/metabolismo , Obesidad Mórbida/cirugía , Grasa Subcutánea Abdominal/metabolismo , Pérdida de Peso , Adulto , Cirugía Bariátrica , Femenino , Humanos , Obesidad Mórbida/metabolismo , Salud de la MujerRESUMEN
Molecular plasticity controls enzymatic activity: the native fold of a protein in a given environment is normally unique and at a global free-energy minimum. Some proteins, however, spontaneously undergo substantial fold switching to reversibly transit between defined conformers, the "metamorphic" proteins. Here, we present a minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, which reversibly transits between several different states of defined three-dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition. The latter is triggered by sequestering the competent conformation in incompetent but structured dimers, tetramers, and octamers. This system, which is compatible with a discrete multifunnel energy landscape, affords a switch that provides a reversible mechanism of control of catalytic activity unique in nature.
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Metaloproteasas/química , Methanocaldococcus/enzimología , Metaloproteasas/metabolismo , Methanocaldococcus/química , Methanocaldococcus/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , TermodinámicaRESUMEN
SCOPE: To determine whether the insulin resistance that exists in metabolic syndrome (MetS) patients is modulated by dietary fat composition. METHODS AND RESULTS: Seventy-five patients were randomly assigned to one of four diets for 12 wk: high-saturated fatty acids (HSFAs), high-MUFA (HMUFA), and two low-fat, high-complex carbohydrate (LFHCC) diets supplemented with long-chain n-3 (LFHCC n-3) PUFA or placebo. At the end of intervention, the LFHCC n-3 diet reduced plasma insulin, homeostasis model assessment of insulin resistance, and nonsterified fatty acid concentration (p < 0.05) as compared to baseline Spanish habitual (BSH) diet. Subcutaneous white adipose tissue (WAT) analysis revealed decreased EH-domain containing-2 mRNA levels and increased cbl-associated protein gene expression with the LFHCC n-3 compared to HSFA and HMUFA diets, respectively (p < 0.05). Moreover, the LFHCC n-3 decreased gene expression of glyceraldehyde-3-phosphate dehydrogenase with respect to HMUFA and BSH diets (p < 0.05). Finally, proteomic characterization of subcutaneous WAT identified three proteins of glucose metabolism downregulated by the LFHCC n-3 diet, including annexin A2. RT-PCR analysis confirmed the decrease of annexin A2 (p = 0.027) after this diet. CONCLUSION: Our data suggest that the LFHCC n-3 diet reduces systemic insulin resistance and improves insulin signaling in subcutaneous WAT of MetS patients compared to HSFA and BSH diets consumption.
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Tejido Adiposo Blanco/metabolismo , Dieta , Grasas de la Dieta/administración & dosificación , Resistencia a la Insulina , Síndrome Metabólico/metabolismo , Grasa Subcutánea/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Presión Sanguínea , Índice de Masa Corporal , Carbohidratos de la Dieta/administración & dosificación , Ácidos Grasos Monoinsaturados , Ácidos Grasos Insaturados/administración & dosificación , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Insulina/sangre , Estilo de Vida , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Satiety and appetite signaling are accomplished by circulating peptide hormones. These peptide hormones require processing from larger precursors to become bioactive, often by the proprotein convertase 1/3 (PC1/3). Several subcellular maturation steps are necessary for PC1/3 to achieve its optimal enzymatic activity. Certain PC1/3 variants found in the general population slightly attenuate its enzymatic activity and are associated with obesity and diabetes. However, mutations that increase PC1/3 activity and/or affect its specificity could also have physiological consequences. We here present data showing that the known human Ser357Gly PC1/3 mutant (PC1/3(S357G)) represents a PC1/3 hypermorph. Conditioned media from human embryonic kidney-293 cells transfected with PC1/3(WT) and PC1/3(S357G) were collected and enzymatic activity characterized. PC1/3(S357G) exhibited a lower calcium dependence; a higher pH optimum (neutral); and a higher resistance to peptide inhibitors than the wild-type enzyme. PC1/3(S357G) exhibited increased cleavage to the C-terminally truncated form, and kinetic parameters of the full-length and truncated mutant enzymes were also altered. Lastly, the S357G mutation broadened the specificity of the enzyme; we detected PC2-like specificity on the substrate proCART, the precursor of the cocaine- and amphetamine regulated transcript neuropeptide known to be associated with obesity. The production of another anorexigenic peptide normally synthesized only by PC2, αMSH, was increased when proopiomelanocortin was coexpressed with PC1/3(S357G). Considering the aberrant enzymatic profile of PC1/3(S357G), we hypothesize that this enzyme possesses unusual processing activity that may significantly change the profile of circulating peptide hormones.
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Mutación Missense , Proproteína Convertasa 1/química , Proproteína Convertasa 1/genética , Secuencia de Aminoácidos , Estabilidad de Enzimas , Glicina/genética , Glicina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Proproteína Convertasa 1/metabolismo , Estructura Terciaria de Proteína , Serina/genética , Serina/metabolismoRESUMEN
Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.
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Mutación , Obesidad/genética , Proproteína Convertasa 1/genética , Animales , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Femenino , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Immunoblotting , Insulina/biosíntesis , Islotes Pancreáticos/metabolismo , Cinética , Masculino , Ratones , Microscopía Confocal , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Obesidad/metabolismo , Proproteína Convertasa 1/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Proteolisis , Ubiquitina/metabolismoRESUMEN
Mitochondria play a key role as major regulators of cellular energy homeostasis, but in the context of mitochondrial dysfunction, mitochondria may generate reactive oxidative species and induce cellular apoptosis. Indeed, altered mitochondrial status has been linked to the pathogenesis of several metabolic disorders and specially disorders related to insulin resistance, such as obesity, type 2 diabetes, and other comorbidities comprising the metabolic syndrome. In the present review, we summarize information from various mitochondrial proteomic studies of insulin-sensitive tissues under different metabolic states. To that end, we first focus our attention on the pancreas, as mitochondrial malfunction has been shown to contribute to beta cell failure and impaired insulin release. Furthermore, proteomic studies of mitochondria obtained from liver, muscle, and adipose tissue are summarized, as these tissues constitute the primary insulin target metabolic tissues. Since recent advances in proteomic techniques have exposed the importance of PTMs in the development of metabolic disease, we also present information on specific PTMs that may directly affect mitochondria during the pathogenesis of metabolic disease. Specifically, mitochondrial protein acetylation, phosphorylation, and other PTMs related to oxidative damage, such as nitrosylation and carbonylation, are discussed.
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Enfermedades Metabólicas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteómica/métodos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Humanos , Insulina/metabolismo , Enfermedades Metabólicas/patología , Mitocondrias/patología , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Estrés Oxidativo , Procesamiento Proteico-PostraduccionalRESUMEN
Obesity is dramatically increasing virtually worldwide, which has been linked to the rising prevalence of metabolic syndrome. Excess fat accumulation causes severe alterations in adipose tissue function. Actually, adipose tissue is now recognized as a major endocrine and secretory organ that releases a wide variety of signaling molecules (hormones, growth factors, cytokines, chemokines, etc.), the adipokines, which play central roles in the regulation of energy metabolism and homeostasis, immunity and inflammation. In addition, adipose tissue is no longer regarded as a passive lipid storage site but as a highly dynamic energy depot which stores excess energy during periods of positive energy balance and mobilizes it in periods of nutrient deficiency in a tightly regulated manner. Altered lipid release and adipokine production and signaling, as occurs in obesity, are linked to insulin resistance and the associated comorbidities of metabolic syndrome (dyslipidemia, hypertension), which confer an increased risk for the development of type 2 diabetes and cardiovascular disease. Here we summarize current knowledge on adipose tissue and review the contribution of novel techniques and experimental approaches in adipobiology to the identification of novel biomarkers and potential targets for dietary or pharmacological intervention to prevent and treat adipose tissue-associated diseases.
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Adipoquinas/sangre , Tejido Adiposo/metabolismo , Síndrome Metabólico/sangre , Síndrome Metabólico/terapia , Animales , Metabolismo Energético/fisiología , Humanos , Resistencia a la Insulina/fisiología , Síndrome Metabólico/epidemiología , Obesidad/sangre , Obesidad/epidemiología , Obesidad/terapiaRESUMEN
The deposition of fibrillated human islet ß-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.