Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR-ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.he aryl hydrocarbon receptor (AHR) belongs to the PER-ARNT-SIM (PAS) family transcription factor that mediates broad responses to cellular and environmental cues. The AHR has been shown to be activated by diverse environmental toxicants and endogenous ligands, and play an important role in adaptive metabolism, dioxin toxicity, and normal vascular and immune development (1, 2), ever since it was identified four decades ago for mediating metabolic responses to aryl hydrocarbon toxicants (3, 4) and the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (5). The AHR was more recently found to mediate diverse cellular and physiological responses and likely respond to unknown endogenous AHR ligands (1, 2, 6-8). Developmentally, the AHR plays a role in the normal development and function of both the vascular and immune systems (9-12), and has close links to cancer, metabolic, immune, and cardiovascular diseases (13-18).Intense efforts in the past four decades have yielded important insights into the molecular processes governing AHR signaling. Newly synthesized AHR is located in cytosol and associated with the chaperones Hsp90 (19), P23 (20, 21), and AHR associated protein 9 (ARA9, also known as XAP2 or AIP) (22-24). Binding of ligands induces conformational changes in the AHR that lead to exposure of nuclear localization sequences (NLS) (25, 26). Following nuclear translocation, the AHR exchanges chaperones for a transcription partner, ARNT (1) and the AHR-ARNT heterodimer binds near the promoters of target genes at dioxin-response element (DRE...

Background. Total laparoscopic hysterectomy (TLH) causes various types of postoperative pain, and the pain pattern has not been evaluated in detail to date. This prospective observational study investigated the types of postoperative pain, intensity in the course of time, and pain characteristics during the first postoperative 72 hr after TLH.Methods. Sixty four female patients undergoing TLH were enrolled, which finally 50 patients were included for the data analyses. The locations of pain included overall pain, abdominal visceral and incisional pains, shoulder pain, and perineal pain. Assessments were made at rest and in motion, and pain level was scored with the use of the 100 mm visual analog scale. The pain was assessed at baseline, and at postoperative 30 min, 1 hr, 3 hr, 6 hr, 24 hr, 48 hr, and 72 hr.Results. Overall, visceral, and incisional pains were most intense on the day of operation and then decreased following surgery. In contrast, shoulder pain gradually increased, peaking at postoperative 24 hr. Shoulder pain developed in 90% of all patients (44/50). It was not more aggravated in motion than at rest, in comparison with other pains, and right shoulder pain was more severe than left shoulder pain (p=0.006). In addition, the preoperative exercise habit of patients increased the threshold of shoulder pain. Most patients (46/50) had perineal pain, which was more severe than abdominal pain in approximately 30% of patients (17/50).Conclusion. Pain after TLH showed considerably different duration, severity, and characteristics, compared with other laparoscopic procedures. Shoulder pain was most intense at postoperative 24 hr, and the intensity was associated with the prior exercise habit of patients and the high level of analgesic request.

The prokaryotic global transcription factor CRP has been considered to be an ideal model for in-depth study of both the allostery of the protein and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and cGMP. Here, atomic details from the crystal structures of two inactive CRP species, an apo form and a cGMP-bound form, in comparison with a known active conformation, the cAMP-CRP complex, provide macroscopic and microscopic insights into CRP allostery, which is coupled to specific discrimination between the two effectors. The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including secondary-structure formation. The observed conformational asymmetries underlie a negative cooperativity in the sequential binding of cyclic nucleotides and a stepwise manner of binding with discrimination between the effector molecules. Additionally, the finding that cGMP, which is specifically recognized in a syn conformation, induces an inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP signalling could be widely utilized in prokaryotes, including in aggressive inhibition of CRP-like proteins.