The penultimate step of proteasomal ATPase assembly is mediated by a switch dependent on the chaperone Nas2.

The proteasome holoenzyme is a complex molecular machine that degrades most proteins. In the proteasome holoenzyme, six distinct ATPase subunits (Rpt1 through Rpt6) enable protein degradation by injecting protein substrates into it. Individual Rpt subunits assemble into a heterohexameric "Rpt ring" in a stepwise manner, by binding to their cognate chaperones. Completion of the heterohexameric Rpt ring correlates with release of a specific chaperone, Nas2; however, it is unclear whether and how this event may ensure proper Rpt ring assembly. Here, we examined the action of Nas2 by capturing the poorly characterized penultimate step of heterohexameric Rpt ring assembly. For this, we used a heterologous Escherichia coli system coexpressing all Rpt subunits and assembly chaperones as well as Saccharomyces cerevisiae to track Nas2 actions during endogenous Rpt ring assembly. We show that Nas2 uses steric hindrance to block premature progression of the penultimate step into the final step of Rpt ring assembly. Importantly, Nas2 can activate an assembly checkpoint via its steric activity, when the last ATPase subunit, Rpt1, cannot be added in a timely manner. This checkpoint can be relieved via Nas2 release, when Nas2 recognizes proper addition of Rpt1 to one side of its cognate Rpt5, and ATP hydrolysis by Rpt4 on the other side of Rpt5, allowing completion of Rpt ring assembly. Our findings reveal dual criteria for Nas2 release, as a mechanism to ensure both the composition and functional competence of a newly assembled proteasomal ATPase, to generate the proteasome holoenzyme.

In utero exposure to phthalate downregulates critical genes in Leydig cells of F1 male progeny.

Phthalates are the largest group of environmental pollutants and are considered toxicant to the endocrine system. The present study was aimed to test the effect of in utero exposure of di(2-ethylhexyl)phthalate (DEHP) on Leydig cell steroidogenesis in F1 male offspring's. Pregnant dams were oral gavaged with different doses (1, 10, and 100 mg/kg/day) of DEHP or olive oil during gestational Day 9-21. Serum testosterone (T) and estradiol (E2) levels were significantly reduced in male offspring at 60 days of age. Our results also demonstrate a coordinate, dose-dependent disruption of genes involved in steroidogenesis. The gene expression of StAR, Cyp11a1, 3ß-HSD, 17ß-HSD, 5α-reductase and cytochrome P450 19a1 (or) aromatase (Cyp-19) were significantly decreased. The transcription factors like steroidogenic factor-1 (SF-1) and specific protein-1 (Sp-1) showed a significant decrease in 10 and 100 mg DEHP treatment group. DNA methylation analysis using bisulfite specific-methylation PCR shows hypermethylation in the SF-1 and Sp-1 promoter regions. Further to determine whether the DEHP-induced methylation changes were associated with increased DNA methyltransferase (Dnmt) levels, we measured the expression levels of Dnmt3a, Dnmt3b, Dnmt1, and Dnmt3l using real-time PCR and Western blot method. The mRNA and protein expressions of Dnmt3a, Dnmt3b, and Dnmt1 were stimulated in 10 and 100 mg DEHP treatment groups, whereas no significant change was seen in Dnmt3l expression, suggesting that increased Dnmt3a/b, Dnmt1 may cause DNA hypermethylation in testicular Leydig cells. Overall, these data suggest that gestational exposure to DEHP affects adult testicular function via altered methylation patterns.

Protective role of quercetin on polychlorinated biphenyls (Aroclor-1254) induced oxidative stress and apoptosis in liver of adult male rats.

The present study aims to investigate the protective effect of quercetin against Aroclor-1254-induced hepatotoxicity in rats. Male Wistar rats were grouped into Group I control received vehicle (corn oil; 1 mL/kg bwt); Group II quercetin alone (50 mg/kg bwt/day orally); Group III Aroclor-1254 (2 mg/kg bwt/day intraperitoneally); Group IV Aroclor-1254 + quercetin treated for 30 days. The Aroclor-1254 treatment caused significant alteration in the biochemical parameters (hydrogen peroxide, lipid peroxidation, reduced glutathione levels, and alkaline phosphatase activity). The expressions of apoptotic and antiapoptotic proteins and the liver histology of Aroclor-1254-exposed rats showed cytoplasmic degeneration along with infiltration of polymorphonuclear cells. Whereas simultaneous treatment with quercetin normalized all the biochemical parameters, consequently it inhibited apoptosis mediated by Aroclor-1254 by downregulating aryl hydrocarbon receptor, p53 and apoptotic protein (Bax, caspase-9, caspase-3) and upregulating the antiapoptotic protein (Bcl-2) expression patterns; thereby, quercetin reduces alteration in hepatocellular morphology. Thus quercetin exhibited hepatoprotective effect.

Assembly checkpoint of the proteasome regulatory particle is activated by coordinated actions of proteasomal ATPase chaperones.

The proteasome holoenzyme regulates the cellular proteome via degrading most proteins. In its 19-subunit regulatory particle (RP), a heterohexameric ATPase enables protein degradation by injecting protein substrates into the core peptidase. RP assembly utilizes "checkpoints," where multiple dedicated chaperones bind to specific ATPase subunits and control the addition of other subunits. Here, we find that the RP assembly checkpoint relies on two common features of the chaperones. Individual chaperones can distinguish an RP, in which their cognate ATPase persists in the ATP-bound state. Chaperones then together modulate ATPase activity to facilitate RP subunit rearrangements for switching to an active, substrate-processing state in the resulting proteasome holoenzyme. Thus, chaperones may sense ATP binding and hydrolysis as a readout for the quality of the RP complex to generate a functional proteasome holoenzyme. Our findings provide a basis to potentially exploit the assembly checkpoints in situations with known deregulation of proteasomal ATPase chaperones.