CHK2-mediated regulation of PARP1 in oxidative DNA damage response.

Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor, which upon activation, recruits downstream proteins by poly(ADP-ribosyl)ation (PARylation). However, it remains largely unclear how PARP1 activity is regulated. Interestingly, the data obtained through this study revealed that PARP1 was co-immunoprecipitated with checkpoint kinase 2 (CHK2), and the interaction was increased after oxidative DNA damage. Moreover, CHK2 depletion resulted in a reduction in overall PARylation. To further explore the functional relationship between PARP1 and CHK2, this study employed H2O2 to induce an oxidative DNA damage response in cells. Here, we showed that CHK2 and PARP1 interact in vitro and in vivo through the CHK2 SCD domain and the PARP1 BRCT domain. Furthermore, CHK2 stimulates the PARylation activity of PARP1 through CHK2-dependent phosphorylation. Consequently, the impaired repair associated with PARP1 depletion could be rescued by re-expression of wild-type PARP1 and the phospho-mimic but not the phospho-deficient mutant. Mechanistically, we showed that CHK2-dependent phosphorylation of PARP1 not only regulates its cellular localization but also promotes its catalytic activity and its interaction with XRCC1. These findings indicate that CHK2 exerts a multifaceted impact on PARP1 in response to oxidative stress to facilitate DNA repair and to maintain cell survival.

Hsp90 mediates the crosstalk between galactose metabolism and cell morphology pathways in yeast.

Galactose metabolism in the yeast Saccharomyces cerevisiae is carried out by a specialized GAL pathway consisting of structural and regulatory proteins. It is known that cells with unbalanced Gal proteins accumulate toxic metabolic intermediates and exhibit severe growth defects. Recently, we found that the molecular chaperone Hsp90 controls the abundance of multiple Gal proteins, possibly to prevent these defects. Hsp90 regulates various cellular processes including cell morphology in response to environmental cues. Yeast cells are known to resort to filamentous growth upon exposure to galactose or other environmental stresses. Our previous and current findings support the "Hsp90 titration model" of Hsp90 buffering, which links the cell morphology and galactose pathways. Our results suggest that, when a large proportion of Hsp90 molecules are used to help Gal proteins, the Hsp90 client proteins in cell morphology pathways are left unattended, leading to filamentous growth. It remains unclear whether this phenomenon serves any biological function or simply reflects a cellular constraint. Nonetheless, it provides an alternative explanation why the GAL pathway is degenerated in some yeast species.

Hsp90 Maintains Proteostasis of the Galactose Utilization Pathway To Prevent Cell Lethality.

Hsp90 is a molecular chaperone that aids in the folding of its metastable client proteins. Past studies have shown that it can exert a strong impact on some cellular pathways by controlling key regulators. However, it is unknown whether several components of a single pathway are collectively regulated by Hsp90. Here, we observe that Hsp90 influences the protein abundance of multiple Gal proteins and the efficiency of galactose utilization even after the galactose utilization pathway (GAL pathway) is fully induced. The effect of Hsp90 on Gal proteins is not at the transcriptional level. Moreover, Gal1 is found to physically interact with Hsp90, and its stability is reduced in low-Hsp90 cells. When Hsp90 is compromised, several Gal proteins form protein aggregates that colocalize with the disaggregase Hsp104. These results suggest that Gal1 and other Gal proteins are probably the clients of Hsp90. An unbalanced GAL pathway has been known to cause fatal growth arrest due to accumulation of toxic galactose metabolic intermediates. It is likely that Hsp90 chaperones multiple Gal proteins to maintain proteostasis and prevent cell lethality especially in a fluctuating environment.

The Hsp90-dependent proteome is conserved and enriched for hub proteins with high levels of protein-protein connectivity.

Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein-protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the "stable isotope labeling by amino acids in cell culture" method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.