Proteasome Activator Enhances Survival of Huntington's Disease Neuronal Model Cells

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28γ, but not subunit S5a, recovered proteasome function in the HD cells. PA28γ also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells

A Regulatory Mechanism Involving TBP-1/Tat-Binding Protein 1 and Akt/PKB in the Control of Cell Proliferation

TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells

Tyrosine Nitration of PA700 Links Proteasome Activation to Endothelial Dysfunction in Mouse Models with Cardiovascular Risk Factors

Oxidative stress is believed to cause endothelial dysfunction, an early event and a hallmark in cardiovascular diseases (CVD) including hypertension, diabetes, and dyslipidemia. However, the targets for oxidative stress-mediated endothelial dysfunction in CVD have not been completely elucidated. Here we report that 26S proteasome activation by peroxynitrite (ONOO−) is a common pathway for endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia. Endothelial function, assayed by acetylcholine-induced vasorelaxation, was impaired in parallel with significantly increased 26S proteasome activity in aortic homogenates from streptozotocin (STZ)-induced type I diabetic mice, angiotensin-infused hypertensive mice, and high fat-diets -fed LDL receptor knockout (LDLr−/−) mice. The elevated 26S proteasome activities were accompanied by ONOO−-mediated PA700/S10B nitration and increased 26S proteasome assembly and caused accelerated degradation of molecules (such as GTPCH I and thioredoxin) essential to endothelial homeostasis. Pharmacological (administration of MG132) or genetic inhibition (siRNA knockdown of PA700/S10B) of the 26S proteasome blocked the degradation of the vascular protective molecules and ablated endothelial dysfunction induced by diabetes, hypertension, and western diet feeding. Taken together, these results suggest that 26S proteasome activation by ONOO−-induced PA700/S10B tyrosine nitration is a common route for endothelial dysfunction seen in mouse models of hypertension, diabetes, and dyslipidemia

Genetic determinants of risk in pulmonary arterial hypertension:international genome-wide association studies and meta-analysis

Background: Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes. Methods: We did two separate genome-wide association studies (GWAS) and a meta-analysis of pulmonary arterial hypertension. These GWAS used data from four international case-control studies across 11 744 individuals with European ancestry (including 2085 patients). One GWAS used genotypes from 5895 whole-genome sequences and the other GWAS used genotyping array data from an additional 5849 individuals. Cross-validation of loci reaching genome-wide significance was sought by meta-analysis. Conditional analysis corrected for the most significant variants at each locus was used to resolve signals for multiple associations. We functionally annotated associated variants and tested associations with duration of survival. All-cause mortality was the primary endpoint in survival analyses. Findings: A locus near SOX17 (rs10103692, odds ratio 1·80 [95% CI 1·55–2·08], p=5·13 × 10 –15 ) and a second locus in HLA-DPA1 and HLA-DPB1 (collectively referred to as HLA-DPA1/DPB1 here; rs2856830, 1·56 [1·42–1·71], p=7·65 × 10 –20 ) within the class II MHC region were associated with pulmonary arterial hypertension. The SOX17 locus had two independent signals associated with pulmonary arterial hypertension (rs13266183, 1·36 [1·25–1·48], p=1·69 × 10 –12 ; and rs10103692). Functional and epigenomic data indicate that the risk variants near SOX17 alter gene regulation via an enhancer active in endothelial cells. Pulmonary arterial hypertension risk variants determined haplotype-specific enhancer activity, and CRISPR-mediated inhibition of the enhancer reduced SOX17 expression. The HLA-DPA1/DPB1 rs2856830 genotype was strongly associated with survival. Median survival from diagnosis in patients with pulmonary arterial hypertension with the C/C homozygous genotype was double (13·50 years [95% CI 12·07 to &gt;13·50]) that of those with the T/T genotype (6·97 years [6·02–8·05]), despite similar baseline disease severity. Interpretation: This is the first study to report that common genetic variation at loci in an enhancer near SOX17 and in HLA-DPA1/DPB1 is associated with pulmonary arterial hypertension. Impairment of SOX17 function might be more common in pulmonary arterial hypertension than suggested by rare mutations in SOX17. Further studies are needed to confirm the association between HLA typing or rs2856830 genotyping and survival, and to determine whether HLA typing or rs2856830 genotyping improves risk stratification in clinical practice or trials. Funding: UK NIHR, BHF, UK MRC, Dinosaur Trust, NIH/NHLBI, ERS, EMBO, Wellcome Trust, EU, AHA, ACClinPharm, Netherlands CVRI, Dutch Heart Foundation, Dutch Federation of UMC, Netherlands OHRD and RNAS, German DFG, German BMBF, APH Paris, INSERM, Université Paris-Sud, and French ANR. </p

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle