The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for ...degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.
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•Hsc70/Hsp70 engage in novel bipartite binding mode with CHIP•Hsp70-lid interaction with CHIP is required for ubiquitination of Hsp70 clients•TPR:lid-tail structure allows modeling of full-length Hsp70:CHIP complexes•Phosphorylation or methylation of Hsp70-lid residues regulate interaction with CHIP
Zhang et al. report a novel interaction between Hsp70 and CHIP. This interaction allows for full-length models of the CHIP/Hsp70 complex to be assembled, predicts how Hsp70 posttranslational modifications regulate ubiquitination, and suggests a new mode of chaperone/cochaperone interactions.
Mortalin, a member of the Hsp70‐family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe‐S cluster protein biogenesis, ...mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT‐077. Like other Hsp70‐family members, Mortalin consists of a nucleotide‐binding domain (NBD) and a substrate‐binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide‐binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease‐associated mutation is located on the Mortalin‐NBD surface and may contribute to Mortalin aggregation. We present structure‐based models for how the Mortalin‐NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT‐077. Our structure may contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.
Interactive Figure 1; Interactive Figure 2 | PDB Code(s): 4KBO
RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and ...scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.
Monogenic IFN-mediated autoinflammatory diseases present in infancy with systemic inflammation, an IFN response gene signature, inflammatory organ damage, and high mortality. We used the JAK ...inhibitor baricitinib, with IFN-blocking activity in vitro, to ameliorate disease.
Between October 2011 and February 2017, 10 patients with CANDLE (chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures), 4 patients with SAVI (stimulator of IFN genes-associated STING-associated vasculopathy with onset in infancy), and 4 patients with other interferonopathies were enrolled in an expanded access program. The patients underwent dose escalation, and the benefit was assessed by reductions in daily disease symptoms and corticosteroid requirement. Quality of life, organ inflammation, changes in IFN-induced biomarkers, and safety were longitudinally assessed.
Eighteen patients were treated for a mean duration of 3.0 years (1.5-4.9 years). The median daily symptom score decreased from 1.3 (interquartile range IQR, 0.93-1.78) to 0.25 (IQR, 0.1-0.63) (P < 0.0001). In 14 patients receiving corticosteroids at baseline, daily prednisone doses decreased from 0.44 mg/kg/day (IQR, 0.31-1.09) to 0.11 mg/kg/day (IQR, 0.02-0.24) (P < 0.01), and 5 of 10 patients with CANDLE achieved lasting clinical remission. The patients' quality of life and height and bone mineral density Z-scores significantly improved, and their IFN biomarkers decreased. Three patients, two of whom had genetically undefined conditions, discontinued treatment because of lack of efficacy, and one CANDLE patient discontinued treatment because of BK viremia and azotemia. The most common adverse events were upper respiratory infections, gastroenteritis, and BK viruria and viremia.
Upon baricitinib treatment, clinical manifestations and inflammatory and IFN biomarkers improved in patients with the monogenic interferonopathies CANDLE, SAVI, and other interferonopathies. Monitoring safety and efficacy is important in benefit-risk assessment.
ClinicalTrials.gov NCT01724580 and NCT02974595.
This research was supported by the Intramural Research Program of the NIH, NIAID, and NIAMS. Baricitinib was provided by Eli Lilly and Company, which is the sponsor of the expanded access program for this drug.
Highlights•UV disinfection of trypsin is feasible, with minimal impact on trypsin function. •UV doses higher than what is required for disinfection diminish trypsin function. •High UV doses result in ...disulfide breakage, fragmentation, and structural changes. •Not all proteins have the same resistance to UV irradiation.
The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the ...treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule ...inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
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•Moderate UV can disinfect cell culture media without damage to the media.•H2O2 generated by high UV was investigated as an initiator of deleterious reactions.•UV causes changes ...beyond what are caused by H2O2 formation.•H2O2 in media only caused changes in pyruvate, acetate, sarcosine and formate.•Catalase addition prevented changes in pyruvate, acetate, sarcosine and formate.
Ultraviolet (UV) irradiation is being considered for protection against viral contamination in cell culture media. Hydrogen peroxide (H2O2) produced by UV irradiation has been suggested as the cause for poor cell growth in irradiated media, but this hypothesis has not been carefully evaluated. The impact of H2O2 on Chinese Hamster Ovary (CHO) cell culture medium was compared with the impact of UV irradiation. Media composition was analyzed via nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LCMS), and cell growth in treated media was also evaluated. Although addition of H2O2 to medium caused significant changes in pyruvate, formate, acetate, and sarcosine concentrations, there was less effect on CHO cell growth compared with irradiation. UV irradiation caused other changes in composition that did not occur as a result of H2O2 addition. Catalase inhibited the effects of adding H2O2 to the media, but catalase added before irradiation did not affect most irradiation-induced changes, even though catalase retained activity. In conclusion we found that while H2O2, which can be generated as a result of UV-irradiation, may be the cause of some changes in medium composition, it does not directly account for impaired CHO cell growth after high UV doses.
Abstract The comparative effects of aerobic and resistance exercise on triglyceride-rich lipoproteins including remnant lipoproteins are controversial. This study examined exercise effect on ...remnant-like lipoprotein particle cholesterol (RLP-C) in type 2 diabetes. Participants were randomized to control (Control), aerobic (Aerobic), resistance (Resistance), or both (Combined) exercise groups. Baseline and 6-month fasting RLP-C and apolipoprotein B48 concentrations were measured. Data analysis was on an intention-to-treat basis. At 6 months, RLP-C was lower in all groups; ΔRLP-C mg/dl, (95% confidence interval), Control −3.91, (−6.21 to −1.6), p = 0.001; Aerobic −3.89, (−6.41 to −1.36), p = 0.003, Resistance −7.52, (−9.89 to −5.15), p = 0.0001, Combined −7.50, (−9.87 to −5.13), p = 0.0001. Total triglycerides were significantly lower in Resistance and Combined groups only; −17.7 mg/dl (−32.8 to −2.7), p = 0.02 and −27.5 (−42.5 to −11.5), p = 0.001, respectively. Inter-group comparisons showed no difference in RLP-C change between Aerobic and Control and a significant difference in RLP-C change only where groups incorporating resistance exercise were compared with those without. There was no significant difference in RLP-C change between Resistance and Combined. Inter-group comparisons of total triglycerides change were significant only between Combined and Control. Changes in apolipoprotein B48 were not significant in inter-group comparisons. In conclusion, our data indicate that resistance exercise training, not aerobic, lowers RLP-C in type 2 diabetes. This effect was not revealed by changes in total triglycerides and apolipoprotein B48. The discordance between changes in RLP-C and apolipoprotein B48 in response to resistance exercise may indicate (a) a decrease in VLDL remnant and not chylomicron remnant particle number and/or (b) a depletion of cholesterol in chylomicron and/or VLDL remnants.
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