RESUMEN
Aging is a prominent risk factor for Alzheimer's disease (AD), but the cellular mechanisms underlying neuronal phenotypes remain elusive. Both accumulation of amyloid plaques and neurofibrillary tangles in the brain1 and age-linked organelle deficits2-7 are proposed as causes of AD phenotypes but the relationship between these events is unclear. Here, we address this question using a transdifferentiated neuron (tNeuron) model directly from human dermal fibroblasts. Patient-derived tNeurons retain aging hallmarks and exhibit AD-linked deficits. Quantitative tNeuron proteomic analyses identify aging and AD-linked deficits in proteostasis and organelle homeostasis, particularly affecting endosome-lysosomal components. The proteostasis and lysosomal homeostasis deficits in aged tNeurons are exacerbated in sporadic and familial AD tNeurons, promoting constitutive lysosomal damage and defects in ESCRT-mediated repair. We find deficits in neuronal lysosomal homeostasis lead to inflammatory cytokine secretion, cell death and spontaneous development of Aß and phospho-Tau deposits. These proteotoxic inclusions co-localize with lysosomes and damage markers and resemble inclusions in brain tissue from AD patients and APP-transgenic mice. Supporting the centrality of lysosomal deficits driving AD phenotypes, lysosome-function enhancing compounds reduce AD-associated cytokine secretion and Aß deposits. We conclude that proteostasis and organelle deficits are upstream initiating factors leading to neuronal aging and AD phenotypes.
RESUMEN
The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer's disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-ß inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.
RESUMEN
During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.
Asunto(s)
Células Eritroides/citología , Eritropoyesis/fisiología , Proteínas Ribosómicas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Anemia/genética , Anemia Hipocrómica/genética , Animales , Eritrocitos/citología , Eritrocitos/enzimología , Células Eritroides/enzimología , Eritropoyesis/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteoma/metabolismo , Proteómica , Reticulocitos/citología , Reticulocitos/enzimología , Ribosomas/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Phosphorylation is a common but crucial protein posttranslational modification occurring in virtually all known species. A successful technique for identifying phosphorylation sites is via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition to identification, the introduction of stable isotopes allows for LC-MS based quantification of thousands of phosphorylation sites. Historically, stable isotope labeling by amino acids in cell culture (SILAC) has been the preferred method for introducing stable isotopes for quantification. SILAC is not well suited, however, for quantitative proteomics in larger animals. The introduction of stable isotope instead by reductive dimethylation is an alternative for performing quantitative proteomics in animal tissues. Here we present an improved reductive dimethylation protocol and demonstrate the application of this method in the analysis of the fasted vs. re-fed mouse liver phosphoproteome. In our analysis, greater than 8500 sites were identified from â¼2700 phosphoproteins. Nearly 7400 phosphorylation events from â¼2300 phosphoproteins were reliably quantified. Using a 2-fold change as a cutoff, 390 phosphorylation sites were found to change between fasted and re-fed mice, many of which may have interesting biological interpretations.
Asunto(s)
Hígado/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Proteómica/métodos , Secuencias de Aminoácidos , Animales , Deuterio , Dieta , Ayuno , Expresión Génica , Concentración de Iones de Hidrógeno , Marcaje Isotópico , Hígado/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Espectrometría de Masas en TándemRESUMEN
FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4--homolog of mammalian SIRT6 and SIRT7 proteins--promotes DAF-16-dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Sirtuinas , Factores de Transcripción , Acetilación , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Factores de Transcripción Forkhead , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Histona Acetiltransferasas/metabolismo , Longevidad/genética , Longevidad/fisiología , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Transducción de Señal , Sirtuinas/genética , Sirtuinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and ß isoforms in the brain. Although CaMKIIα functions have been elucidated, the isoform-specific catalytic functions of CaMKIIß have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIß operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKIIß to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20-anaphase promoting complex) as a centrosomal substrate of CaMKIIß. CaMKIIß phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKIIß that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Centrosoma/fisiología , Cerebelo/citología , Dendritas/fisiología , Neuronas/citología , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Centrosoma/ultraestructura , Chlorocebus aethiops , Proteínas del Citoesqueleto/metabolismo , Dendritas/genética , Dendritas/ultraestructura , Electroporación/métodos , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Inmunoprecipitación , Espectrometría de Masas , Microscopía Confocal , Microscopía Electrónica de Transmisión/métodos , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Interferencia de ARN/fisiología , Ratas , Transducción de Señal/genética , Estadísticas no Paramétricas , Transfección , Ubiquitina/metabolismoRESUMEN
The glyco-isoforms of intact apolipoprotein C3 (ApoC3) were used to probe glycomic changes associated with obesity and recovery following bariatric surgery, liver diseases such as chronic hepatitis C (CHC) and alcoholic liver cirrhosis, as well as severe, multiorgan diseases such as sepsis and graft vs host disease (GVHD). ApoC3 glyco-isoform ratios responded to unique stimuli that did not correlate with serum lipids or with other blood components measured in either a control population or a group of extremely obese individuals. However, glyco-isoform ratios correlated with obesity with a 1.8-fold change among subjects eligible for bariatric surgery relative to a nonobese control population. Bariatric surgery resulted in rapid change of isoform distribution to that of nonobese individuals, after which the distribution was stable in each individual. Although multiple simultaneous factors complicated effector attribution, the isoform ratios of very obese individuals were nearly normal for diabetic individuals on metformin therapy. Glyco-isoform ratios were sensitive to liver diseases such as chronic hepatitis C and alcoholic liver cirrhosis. The correlation coefficient with fibrosis was superior to that of current assays of serum enzyme levels. Diseases of pregnancy that can result in liver damage, HELLP syndrome and pre-eclampsia, did not alter ApoC3 glyco-isoform ratios. Early after umbilical cord blood transplantation the isoform ratios changed and returned to normal in long-term survivors. Larger changes were observed in persons who died. GVHD had little effect. Persons with severe sepsis showed altered ratios. Similar cut-points for mortality (3.5-fold difference from controls) were found for UCBT and sepsis. Similar values characterized liver cirrhosis. Overall, while changes of glyco-isoform ratios occurred in many situations, individual stability of isoform distribution was evident and large changes were limited to high-level disease. If ratio changes associated with obesity are found to document a risk factor for long-term outcomes, the information provided by glyco-isoform ratio changes may provide important, novel information for diagnostic, prognostic and therapy response to metabolic conditions.
Asunto(s)
Apolipoproteína C-III , Cirugía Bariátrica , Glicósidos/química , Enfermedad Injerto contra Huésped , Hepatopatías/sangre , Metformina/uso terapéutico , Obesidad , Sepsis , Adulto , Anciano , Apolipoproteína C-III/sangre , Apolipoproteína C-III/química , Biomarcadores/sangre , Biomarcadores/química , Diabetes Mellitus/tratamiento farmacológico , Femenino , Enfermedad Injerto contra Huésped/sangre , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/cirugía , Embarazo , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Sepsis/sangre , Sepsis/mortalidadRESUMEN
Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.
Asunto(s)
Proteínas Fúngicas/química , Fosfoproteínas/química , Proteoma , Schizosaccharomyces/química , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Fosforilación , Espectrometría de Masas en TándemRESUMEN
A survey of plasma proteins in approximately 1,300 individuals by MALDI-TOF MS resulted in identification of a structural polymorphism of apolipoprotein C1 (ApoC1) that was found only in persons of American Indian or Mexican ancestry. MS/MS analysis revealed that the alteration consisted of a T45S variation. The methyl group of T45 forms part of the lipid-interacting surface of ApoC1. In agreement with an impact on lipid contact, the S45 variant was more susceptible to N-terminal truncation by dipeptidylpeptidase IV in vitro than was the T45 variant. The S45 protein also displayed greater N-terminal truncation (loss of Thr-Pro) in vivo than the T45 variant. The S45 variant also showed preferential distribution to the very-low-density lipoprotein fraction than the T45 protein. These properties indicate a functional effect of the S45 variant and support a role for residue 45 in lipid contact and lipid specificity. Further studies are needed to determine the effects of the variant and its altered N-terminal truncation on the metabolic functions of ApoC1.