Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in <i>Saccharomyces cerevisiae</i>

<div><p>The role of Hsp70 chaperones in yeast prion propagation is well established. Highly conserved Hsp90 chaperones participate in a number of cellular processes, such as client protein maturation, protein degradation, cellular signalling and apoptosis, but little is known about their role in propagation of infectious prion like aggregates. Here, we examine the influence of Hsp90 in the maintenance of yeast prion [URE3] which is a prion form of native protein Ure2, and reveal a previously unknown role of Hsp90 as an important regulator of [URE3] stability. We show that the C-terminal MEEVD pentapeptide motif, but not the client maturation activity of Hsp90, is essential for [URE3] prion stability. In testing deletions of various Hsp90 co-chaperones known to bind this motif, we find the immunophilin homolog Cpr7 is essential for [URE3] propagation. We show that Cpr7 interacts with Ure2 and enhances its fibrillation. The requirement of Cpr7 is specific for [URE3] as its deletion does not antagonize both strong and weak variant of another yeast prion [<i>PSI</i>^<i>+</i>], suggesting a distinct role of the Hsp90 co-chaperone with different yeast prions. Our data show that, similar to the Hsp70 family, the Hsp90 chaperones also influence yeast prion maintenance, and that immunophilins could regulate protein multimerization independently of their activity as peptidyl-prolyl isomerases.</p></div

The loss of [URE3] upon Cpr7 deletion is not due to altered expression of other major chaperones.

<p>(A) Yeast lysates from wild type (wt) [URE3], wild type [ure-o] and <i>cpr7Δ</i> strains were probed with antibodies against Ydj1, Sse1 and Hsp90. The chaperones were found to be expressed at similar levels in the strains examined. Loading control is same blot stained with amido-black. (B) 5μg (1X) or 10μg (2X) of total lysate protein was loaded into each lane and probed with anti Hsp70 antibodies. As seen, increased Hsp70 level was observed in strain lacking Cpr7 as compared to wt [URE3] or wt [ure-o] strains. (C) SY187(wt) expressing additional Ssa Hsp70 from transformed plasmids pRS315P_SSA2-SSA1/SSA2/SSA3/SSA4 or pRS315 empty vector were spread onto leucine deficient SD solid medium with limiting adenine. As seen by white colony color phenotype of transformants, the increased expression of Ssa Hsp70 supports stable [URE3].</p

Deleting the MEEVD motif of Hsp90 destabilizes [URE3].

<p><b>(A)</b> Effect of deletion and overexpression of Hsp90 on [URE3]. <i>Hsp82Δ</i> (SY295) or <i>Hsc82Δ</i> (SY297) cells grown overnight were subcultured into YPAD medium and grown from O.D._600nm of 0.02 to 1.7. Cells were then plated onto ½ YPD plates and monitored after 3 days of incubation at 30°C. For overexpression studies, SY187 was transformed with plasmid (pRS426P_GPD-His₆-Hsp82 or pRS426P_GPD-His₆-Hsc82) encoding His₆-Hsp82 or His₆-Hsc82 and plated onto uracil deficient solid SD medium with limiting adenine. As seen by white colony color phenotype, no adverse effect on [URE3] stability was observed upon deletion or overexpression of Hsp90 isoforms. <b>(B)</b> The strains constructed as described in Materials and Methods express the indicated Hsp90 isoform as the sole cytosolic Hsp90. Cells were grown from O.D._600nm of 0.02 to 1.7 before plating onto ½ YPD medium. As seen by red colony color phenotype the frequency of [ure-o] colonies increases upon deletion of MEEVD motif.</p

Cns1 complements Cpr7 function required for [URE3] stability.

<p>The <i>cpr7Δ</i> strain was transformed with pRS426P_CNS1-CNS1 or pRS413P_TEF-CPR7, and [URE3] was monitored on ½ YPD plates as described before. All colonies harbouring the Cns1 encoding plasmid show white colony color suggesting functional redundancy of Cns1 and Cpr7 with regard to [URE3] propagation.</p

Cpr7 is required for [URE3] stability.

<p>(A) Effect of deleting genes encoding Hsp90 co-chaperones on prion propagation. Multiple single-knockout strains constructed as described in Materials and Methods were streaked onto a ½ YPD plate and [URE3] was monitored after 3 days of incubation at 30°C and 2 days at room temperature. As seen, the deletion of gene encoding Cpr7 leads to [ure-o] phenotype. (B) Cells were grown in YPAD liquid medium, further grown from O.D._600nm = 0.02 to 1.7 and then plated on ½ YPD. The colonies were then replica-plated onto ½ YPD and adenine deficient plates. As seen, though SY187 [URE3] cells grow normally on adenine deficient medium, only about 2–3% of colonies from <i>cpr7Δ</i> strain grew on solid medium lacking adenine. (C) The effect of Hsp90 co-chaperone deletion on [<i>PSI</i>^<i>+</i>] propagation. Deletion strains of the <i>ade2-1</i> background were assessed as in panel (A). (D) [URE3] diploid strain heterozygous for <i>cpr7Δ</i> (<i>CPR7</i>/<i>cpr7</i>::<i>KanMX</i>) was sporulated and dissected. [URE3] phenotype segregated 2:2 and all <i>cpr7Δ</i> (G418 resistant) spores were found to be [ure-o] as seen by red colony color phenotype.</p

Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in <i>Saccharomyces cerevisiae - Table 1 </i>

<p>Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in <i>Saccharomyces cerevisiae - Table 1 </i></p

Cpr7 tetratricopeptide domain is important for [URE3] prion propagation.

<p>(A) Schematic of domain architecture of Cpr6, Cpr7 and their hybrid proteins. (B) Strains expressing Cpr6, Cpr7 or hybrids in <i>cpr7Δ</i> genetic background were constructed and monitored for [URE3] as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005567#pgen.1005567.g001" target="_blank">Fig 1A</a>. ½ YPD plates were further replicated onto SD lacking histidine (SD, -His) or adenine (SD, -Ade). Shown is the growth after 1day (1D) or 2 days (2D) of incubation at 30°C after replica plating from ½ YPD plate. As seen by white colony color phenotype cells expressing Cpr7, 6PPI/7TPR or 7TPR support stable [URE3]. Red colony color phenotype is seen only in those colonies that either lost the transformed plasmid (due to growth on ½ YPD), harbour empty plasmid or the plasmid encoding for Cpr6, 7PPI/6TPR.</p

Trend of risk factors for cardiovascular diseases among young and middle-aged indians: Insights from a nationally representative survey

Background: India, as a nation is witnessing epidemiological transition, which is taking place across all the states at different level, over past couple of decades. Owing to the long natural history of non-communicable diseases (NCDs), early identification of these risk factors can aid in understanding the distribution and future development of cardiovascular diseases (CVDs). Also, studying the trend of these risk factors over time can help in prediction of burden of various CVDs in future. Thus, the present study aims at understanding the trend of various risk factors for CVDs across rural and urban India, and states.Methods: The present study was conducted using secondary data from the third, fourth and fifth round of the National Family Health Survey (NFHS) conducted in India. The surveys collected data for estimation of burden of the common modifiable risk factors of CVDs including tobacco and alcohol consumption overweight/obesity, raised blood pressure, and raised blood sugar. The analysis for the present study was done among interviewed males and females between 15 and 49 years. The weighted prevalence of these risk factors was computed and binary logistic regression was done to study the predictors for the same.Results: A declining trend of tobacco (29.2% in NFHS 3; 8.1%in NFHS 5) and alcohol consumption (14.2% in NFHS 3; 3.2%in NFHS 5) was observed from 2005 -06 to 2019–21. A rising trend of overall raised blood pressure (11.4% in NFHS 4; 12.2%in NFHS 5), raised blood sugar (6.2% in NFHS 4; 8.5%in NFHS 5), and overweight and obesity (11.4% in NFHS 3; 23.6%in NFHS 5) was observed from the three rounds of the survey. The odds of all the studied risk factors were significantly higher among older age across all the rounds of the survey. Except overweight/obesity, the odds of rest all studied risk factors was found to be higher among males compared to females. Also, higher odds of alcohol consumption, overweight/obesity, raised blood pressure, and raised blood sugar were found among the participants living in urban areas compared to rural areas, across all the rounds of the survey.Conclusion: The present highlights the rising burden of CVD risk factors, including overweight and obesity, raised blood pressure and raised blood sugar, and a declining trend of tobacco and alcohol consumption across the country. The study also highlights the need for in-depth assessment of predictors of these risk factors using longitudinal study designs.</p