RESUMEN

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Asunto(s)

Membrana Basal/química , Membrana Basal/metabolismo , Animales , Membrana Basal/ultraestructura , Humanos , Microscopía de Fuerza Atómica , Proteómica

RESUMEN

Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected in vitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.

Asunto(s)

Ciclopentanos/farmacología , Hipoxia/metabolismo , Prostaglandinas/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Línea Celular , Humanos , Immunoblotting

RESUMEN

Basement membranes (BMs) are extracellular matrix sheets comprising the laminins, type-IV collagens, nidogens, and the heparan sulfate proteoglycans, perlecan, collagen XVIII, and agrin. In intact BMs, BM proteins are physiologically insoluble and partially resistant to proteolytic digestion, making BMs a challenge to study. Here three types of BMs from adult human eyes, the inner limiting membrane (ILM), the retinal vascular BMs, and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS. Peptide and protein identifications were done using MaxQuant. 1129 proteins were identified with a 1% false discovery rate. Data showed that BMs are composed of multiple laminins, collagen IVs, nidogens, and proteoglycans. The dominant laminin family member in all BMs was laminin α5ß2γ1. The dominant collagen IV trimer in lens capsule (LC) and blood vessel (BV) BMs had a chain composition of α1(IV)2, α2 (IV), whereas the dominant collagen IV in the ILM had the α3(IV), α4(IV), α5(IV) chain composition. The data also showed that the ratio of laminin and collagen IVs varied among different BM types: the ratio of collagen IV to the other BM proteins is highest in LC, followed by BV and lowest for the ILM. The data have been deposited to the ProteomeXchange with identifier PXD001025.

RESUMEN

Mitochondrial transcription factor A (TFAM) regulates mitochondrial biogenesis, which is downregulated by extracellular signal-regulated protein kinases (ERK1/2) in cells treated chronically with the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). We utilized mass spectrometry to identify ERK1/2-dependent TFAM phosphorylation sites. Mutation of TFAM at serine 177 to mimic phosphorylation recapitulated the effects of MPP+ in decreasing the binding of TFAM to the light strand promoter, suppressing mitochondrial transcription. Mutant TFAM was unable to affect respiratory function or rescue the effects of MPP+ on respiratory complexes. These data disclose a novel mechanism by which ERK1/2 regulates mitochondrial function through direct phosphorylation of TFAM.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/fisiopatología , Factores de Transcripción/metabolismo , Transcripción Genética , Humanos , Espectrometría de Masas , Mitocondrias/química , Fosforilación , Procesamiento Proteico-Postraduccional

RESUMEN

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive inborn error of metabolism that leads to the impaired mitochondrial fatty acid ß-oxidation of short chain fatty acids. It is heterogeneous in clinical presentation including asymptomatic in most patients identified by newborn screening. Multiple mutations have been identified in patients; however, neither clear genotype-phenotype relationships nor a good correlation between genotype and current biochemical markers for diagnosis has been identified. The definition and pathophysiology of this deficiency remain unclear. To better understand this disorder at a global level, quantitative alterations in the mitochondrial proteome in SCAD deficient mice were examined using a combined proteomics approach: two-dimensional gel difference electrophoresis (2DIGE) followed by protein identification with MALDI-TOF/TOF and iTRAQ labeling followed by nano-LC/MALDI-TOF/TOF. We found broad mitochondrial dysfunction in SCAD deficiency. Changes in the levels of multiple energy metabolism related proteins were identified indicating that a more complex mechanism for development of symptoms may exist. Affected pathways converge on disorders with neurologic symptoms, suggesting that even asymptomatic individuals with SCAD deficiency may be at risk to develop more severe disease. Our results also identified a pattern associated with hepatotoxicity implicated in mitochondrial dysfunction, fatty acid metabolism, decrease of depolarization of mitochondria and mitochondrial membranes, and swelling of mitochondria, demonstrating that SCAD deficiency relates more directly to mitochondrial dysfunction and alteration of fatty acid metabolism. We propose several candidate molecules that may serve as markers for recognition of clinical risk associated with this disorder.

Asunto(s)

Acil-CoA Deshidrogenasa/deficiencia , Hígado/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/análisis , Proteoma/análisis , Acil-CoA Deshidrogenasa/química , Animales , Biomarcadores/metabolismo , Metabolismo Energético , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Errores Innatos del Metabolismo Lipídico/patología , Errores Innatos del Metabolismo Lipídico/fisiopatología , Hígado/fisiopatología , Ratones , Ratones Endogámicos BALB C , Mitocondrias/genética , Oxidación-Reducción

RESUMEN

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.

Asunto(s)

Ácidos Grasos/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Mitocondrias Hepáticas/enzimología , Sirtuina 3/metabolismo , Acetilación , Acil-CoA Deshidrogenasa de Cadena Larga , Animales , Dominio Catalítico/fisiología , Ácidos Grasos/química , Ácidos Grasos/genética , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/genética , Humanos , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/genética , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirtuina 3/química , Sirtuina 3/genética

RESUMEN

Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.

Asunto(s)

Receptores ErbB/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Proteolisis , Ubiquitinación , Secuencia de Aminoácidos , Animales , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Receptores ErbB/química , Humanos , Cinética , Lisosomas/metabolismo , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Sus scrofa , Ubiquitina Tiolesterasa/metabolismo

RESUMEN

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

Asunto(s)

Membrana Basal/química , Membrana Basal/metabolismo , Animales , Membrana Basal/citología , Membrana Basal/ultraestructura , Adhesión Celular , Embrión de Pollo , Colágeno/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Laminina/metabolismo , Ratones , Transporte de Proteínas , Proteoglicanos/metabolismo

RESUMEN

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches.

Asunto(s)

Matriz Extracelular/química , Páncreas/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.

Asunto(s)

Membrana Basal/química , Colágeno Tipo IV/química , Matriz Extracelular/química , Envejecimiento , Animales , Membrana Basal/ultraestructura , Fenómenos Biomecánicos , Adhesión Celular , Técnicas de Cultivo de Célula , Forma de la Célula , Endotelio Vascular/química , Humanos , Ratones , Microscopía Electrónica de Transmisión , Proteoma/análisis , Proteoma/química

RESUMEN

BACKGROUND: The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. RESULTS: We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. CONCLUSION: Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

Asunto(s)

Apoptosis , Histona Acetiltransferasas/metabolismo , Proteínas Oncogénicas v-abl/fisiología , Transcripción Genética , Células 3T3 , Acetilación , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Células Cultivadas , Células HCT116 , Células HeLa , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/fisiología , Humanos , Lisina Acetiltransferasa 5 , Ratones , Proteínas Oncogénicas v-abl/genética , Proteínas Oncogénicas v-abl/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Proto-Oncogenes Mas , ARN Interferente Pequeño/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transcripción Genética/fisiología

RESUMEN

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Electroforesis en Gel Bidimensional/métodos , Paclitaxel/farmacología , Estatmina/fisiología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Microtúbulos/efectos de los fármacos , Mitosis , Índice Mitótico , Proteínas de Neoplasias/efectos de los fármacos , Estatmina/genética , Estatmina/metabolismo , Regulación hacia Arriba

RESUMEN

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ(2)] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ(2) induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ(2) covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate that UCH-L1 function is important in hypoxic neuronal death and that excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1.

Asunto(s)

Hipoxia de la Célula/fisiología , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Prostaglandina D2/análogos & derivados , Ubiquitina Tiolesterasa/metabolismo , Animales , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Hipoxia-Isquemia Encefálica/enzimología , Degeneración Nerviosa/inducido químicamente , Prostaglandina D2/química , Prostaglandina D2/fisiología , Prostaglandina D2/toxicidad , Ratas , Ratas Sprague-Dawley , Transducción Genética/métodos , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética

RESUMEN

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP(+)) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease-associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl-cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.

Asunto(s)

Autofagia/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Bucladesina/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Neuronas/citología , Neuronas/metabolismo , Fagosomas/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Sirolimus/farmacología

RESUMEN

Basement membranes (BMs) are physiologically insoluble extracellular matrix sheets present in all multicellular organisms. They play an important role in providing mechanical strength to tissues and regulating cell behavior. Proteomic analysis of BM proteins is challenged by their high molecular weights and extensive post-translational modifications. Here, we describe the direct analysis of an in vivo BM system using a mass spectrometry (MS) based proteomics approach. Retinal BMs were isolated from embryonic chick eyes. The BM macromolecules were deglycosylated and separated by low percentage gradient SDS PAGE, in-gel digested and analyzed by LC-MS/MS. This identified over 27 extracellular matrix proteins in the retinal BM. A semi-quantitative measure of protein abundance distinguished, nidogens-1 and -2, laminin subunits α1, α5, ß2, and γ1, agrin, collagen XVIII, perlecan, FRAS1 and FREM2 as the most abundant BM protein components. Laminin subunits α3, ß1, γ2, γ3 and collagen IV subunits α5 and α6 were minor constituents. To examine binding interactions that contribute to the stability of the retinal BM, we applied the LC-MS/MS based approach to detect potential BM complexes from the vitreous. Affinity-captured nidogen- and heparin-binding proteins from the vitreous contained >10 and >200 proteins respectively. Comparison of these protein lists with the retinal BM proteome reveals that glycosaminoglycan and nidogen binding interactions play a central role in the internal structure and formation of the retinal BM. In addition, we studied the biomechanical qualities of the retinal BM before and after deglycosylation using atomic force microscopy. These results show that the glycosaminoglycan side chains of the proteoglycans play a dominant role in regulating the thickness and elasticity of the BMs by binding water to the extracellular matrix. To our knowledge, this is the first large-scale investigation of an in vivo BM system using MS-based proteomics.

Asunto(s)

Membrana Basal/química , Membrana Basal/metabolismo , Proteínas de la Matriz Extracelular/análisis , Proteoma/análisis , Proteómica , Retina/metabolismo , Agrina/análisis , Agrina/genética , Agrina/metabolismo , Animales , Fenómenos Biomecánicos , Embrión de Pollo , Colágeno Tipo IV/análisis , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Matriz Extracelular/química , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicosaminoglicanos/análisis , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Proteoglicanos de Heparán Sulfato/análisis , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Glicoproteínas de Membrana/análisis , Microscopía de Fuerza Atómica , Procesamiento Proteico-Postraduccional , Proteoglicanos/análisis , Proteoglicanos/genética , Proteoglicanos/metabolismo , Retina/química , Retina/ultraestructura

RESUMEN

Recent studies have shown a critical function for the ubiquitin-proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non-biased assay to identify fast-turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells. Our characterization showed that UV-induced Rad17 oscillation is mediated by Cdh1/APC, a ubiquitin-protein ligase. Studies using a degradation-resistant Rad17 mutant demonstrated that Rad17 stabilization prevents the termination of checkpoint signalling, which in turn attenuates the cellular re-entry into cell-cycle progression. The findings provide an insight into how the proteolysis of Rad17 by Cdh1/APC regulates the termination of checkpoint signalling and the recovery from genotoxic stress.

Asunto(s)

Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Antígenos CD , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Mutación , Oscilometría/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Asunto(s)

Membrana Basal/fisiología , Animales , Membrana Basal/embriología , Fenómenos Biomecánicos , Lámina Basal de la Coroides/fisiología , Lámina Basal de la Coroides/ultraestructura , Embrión de Pollo , Desecación , Elasticidad , Ratones , Ratones Mutantes , Microscopía de Fuerza Atómica

RESUMEN

Identification of biomarkers could lead to the development of effective screening tests for colorectal cancer. A previous study from our laboratory showed specific alterations of nuclear structure in colon cancer. In an effort to characterize these biomarkers, protein spots were selected from separations made by two-dimensional gel electrophoresis, which were analyzed by mass spectrometry. The sequences obtained from the isolated spots revealed that they have close similarity to creatine kinase B (CKB) isoforms, heterogeneous nuclear ribonucleoprotein F (hnRNP F) and high mobility group box 1 protein (HMGB1) isoforms. To determine the expression of these proteins in colon cancer, expression was studied in 9 tumor and matched adjacent normal pairs, 5 donor colons, 16 polyps, 4 metastatic liver lesions and matched adjacent normal pairs, and 3 liver donors. CKB and hnRNP F were expressed in 78% and 89% of colon tumors, respectively. hnRNP F had a higher frequency of expression than CKB in premalignant polyps. With the establishment of differential expression of the proteins in colon cancer, their subcellular localization was analyzed. The subcellular fractions studied both showed high protein levels of hnRNP F in colon tumors compared with normal colon tissues. Surprisingly, subcellular levels of CKB were decreased in colon tumors, suggesting that the observed high CKB levels in nuclear matrix extracts are caused by the enhanced localization of CKB to the nuclear matrix during colon tumorigenesis. These results suggest an involvement of hnRNP F and CKB in colorectal cancer. Additionally, they suggest that hnRNP F is a potential marker for colorectal cancer progression.

Asunto(s)

Neoplasias del Colon/genética , Forma BB de la Creatina-Quinasa/biosíntesis , Perfilación de la Expresión Génica , Proteínas HMGB/biosíntesis , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/biosíntesis , Neoplasias Hepáticas/secundario , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Anciano , Secuencia de Aminoácidos , Biomarcadores de Tumor/análisis , Estudios de Casos y Controles , Transformación Celular Neoplásica , Neoplasias del Colon/patología , Pólipos del Colon/genética , Pólipos del Colon/patología , Forma BB de la Creatina-Quinasa/análisis , Forma BB de la Creatina-Quinasa/genética , Electroforesis en Gel Bidimensional , Femenino , Proteínas HMGB/análisis , Proteínas HMGB/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/análisis , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Humanos , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular

RESUMEN

Perlecan is a highly conserved heparan sulfate proteoglycan in cartilage and basement membranes. We identified chick perlecan and a 90 KD perlecan fragment in vivo using a newly generated monoclonal antibody. Chick perlecan is, like its human and mouse homologue, a hybrid heparan sulfate/chondroitin sulfate proteoglycan with a core protein of 400 KD. Analysis of the 90 KD fragment by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Capillary LC nano Electrospray Ionization tandem MS (LC nano ESI MS/MS) showed that it belonged to domain IV of the perlecan core protein. We found that full-length perlecan and its domain IV fragment are abundant in embryonic vitreous body and serum. Their expression in vitreous and serum is greatly down-regulated shortly after hatching of the chick. We speculate that the abundance of perlecan in the embryonic circulation and vitreous reflects the ongoing formation of new BMs in the expanding vascular system and the growing retina. In addition, we found that perlecan as a substrate does not support, rather inhibits neurite outgrowth.

Asunto(s)

Proteoglicanos de Heparán Sulfato/sangre , Proteoglicanos de Heparán Sulfato/química , Inmunoglobulinas/química , Cuerpo Vítreo/química , Animales , División Celular , Embrión de Pollo , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Neuritas/fisiología , Estructura Terciaria de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética

RESUMEN

In egg-laying species, such as the chicken, the mode of transport of lipoprotein particles from the capillary plasma to endocytic receptors on the oocyte surface is largely unknown. Here we show by molecular characterization that the large prominent heparan sulfate proteoglycan of extracellular matrices, termed perlecan or HSPG2 (the product of the hspg2 gene), is a component of ovarian follicles that may participate in this process. However, although normally a major HSPG of basement membranes or basal laminae, in chicken follicles, perlecan is absent from the membranous structure between the theca interna and granulosa cell layers, which to date has been considered a bona fide basement membrane. Rather, the protein is localized in the extracellular matrix of theca externa cells, which produce this HSPG. Furthermore, in chicken testes, perlecan is localized in the peritubular spaces but in less organized fashion than the classical basement membrane components, agrin and laminin. All five domains and structural hallmarks of chicken perlecan (4071 residues) have been conserved in its mammalian counterparts. We have produced the recombinant domain II (containing low density lipoprotein (LDL) receptor-like binding repeats) of chicken perlecan and demonstrate its capacity to bind LDL and very low density lipoprotein (VLDL), apolipoprotein B-containing lipoproteins ultimately destined for uptake into oocytes via members of the low density lipoprotein receptor family. Binding to perlecan heparan sulfate side chains may facilitate the interaction of lipoproteins with domain II. Based on the current results and on domain-domain interactions revealed by recent ultrastructural investigations of the LDL receptor, nidogen, and laminin (Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358 and Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974), we propose a novel role of perlecan in mediating plasma-to-oocyte surface transport of VLDL particles.

Asunto(s)

Matriz Extracelular/metabolismo , Proteoglicanos de Heparán Sulfato/química , Folículo Ovárico/metabolismo , Animales , Pollos , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Proteoglicanos de Heparán Sulfato/metabolismo , Lipoproteínas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína