Hamannnicolajsen5213

These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHLHIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. Ac-PHSCN-NH2 supplier While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230-270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.