Cell-type-specific metabolic labeling of nascent proteomes in vivo

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Papel del proteasoma en la proteostasis de [alpha]-sinucleína: implicaciones en enfermedad de Parkinson

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina. Departamento de Bioquímica. Fecha de lectura: 21 de Enero de 201

The regulation of synaptic protein turnover

Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and are modulated by neuronal activity in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but the understanding of protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, as well as the turnover rates of specific neuronal proteins

Effect of internal deletions within the N-terminal region of Nurr1 in its degradation.

<p>HeLa cells were transiently transfected with full-length Nurr1, Nurr1 Δ163–187, Δ163–217 and Δ163–249 as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (A). Protein loading control was assessed by immunobloting with anti-tubulin antibodies (A). (B) Graph shows the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. (C) Cells were also analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for Nurr-1 subcellular localization.</p

Degradation of endogenous Nurr1 in PC12 cells and ectopically expressed Nurr1 in HeLa cells.

<p>(A) PC12 cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies. (B, D and F) HeLa cells were transiently transfected with full-length Nurr1, N-terminal flag-tagged Nurr1 or Nurr1 1–337 as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (B and D). Protein loading control was assessed by immunobloting with anti-tubulin antibodies. (C and E) Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. F, transfected cells were analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for subcellular localization of Nurr1.</p

Effect of the deletions from the N-terminal of Nurr1 on its degradation.

<p>HeLa cells were transiently transfected with full-length Nurr1, Δ1–262, Δ1–161 and Δ1–96 (A) or Nurr1 Δ1–80, Δ1–63, Δ1–43, and Δ1–31 (C) as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (A and C). Protein loading control was assessed by immunobloting with anti-tubulin antibodies. (B and D) Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. (E) Cells were also analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for Nurr-1 subcellular localization of the different Nurr1 constructs as indicated.</p

Effect of treatment of cells with Leptomycin B on the degradation of Nurr1.

<p>HeLa cells were transiently transfected with full-length Nurr1, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) or Leptomycin B for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies Protein loading control was assessed by immunobloting with anti-tubulin antibodies. Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments.</p

Ubquitylation of Nurr1 and Nurr1 Δ1–31.

<p>HeLa cells were co-transfected with Nurr1 full length or Δ1–31 and HA-ubiquitin and either untreated or treated with lactacystin (Lacta) as indicated, cell extracts were immunoprecipatated with anti-HA antibodies, analyzed by SDS-PAGE and immunoblotted with anti-Nurr1 antibodies (A). (B) Direct immunoblot with anti-Nurr1 antibodies of 1/10 of the amount of total cell extracts (shorter exposure than the upper panel) used for immunoprecipitation experiments shown in the upper panel. Protein loading control was assessed by immunobloting with anti-tubulin antibodies.</p

P2X7 receptor inhibition ameliorates ubiquitin–proteasome system dysfunction associated with Alzheimer’s disease

Abstract Background Over recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer’s disease (AD), and the ubiquitin–proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction. Methods To evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R. Results We describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of β5 and β1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival. Conclusions Our work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus