CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.

Necroptosis is an essential pathophysiological process in cerebral ischemia-related diseases. Therefore, targeting necroptosis may prevent cell death and provide a much-needed therapy. Ansiomycin is an inhibitor of protein synthesis which can also activate c-Jun N-terminal kinases. The present study demonstrated that anisomycin attenuated necroptosis by upregulating CHIP (carboxyl terminus of Hsc70-interacting protein) leading to the reduced levels of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) proteins in two in vitro models of cerebral ischemia. Further exploration in this research revealed that losing neither the co-chaperone nor the ubiquitin E3 ligase function of CHIP could abolish its ability to reduce necroptosis. Collectively, this study identifies a novel means of preventing necroptosis in two in vitro models of cerebral ischemia injury through activating the expression of CHIP, and it may provide a potential target for the further study of the disease.

Background: Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is the most common type of autosomal dominant ataxia. Like other neurodegenerative diseases, is characterized by the dysfunction of the protein quality control (PQC) system. The carboxyl terminus of the Hsp70-interacting protein (CHIP), an important component of PQC, participates in the clearance of misfolded proteins to maintain cellular homeostasis. While no cure for SCA3 exists, the disease progresses slowly. Thus, the identification of biomarkers that indicate the severity and prognosis of this disease would be valuable.Methods: In this exploratory case-control study, we quantitatively evaluated the concentrations of CHIP in the sera of 80 patients with SCA3 and 80 age and sex-matched controls, using the enzyme-linked immunosorbent assay (ELISA). CHIP levels in the cerebrospinal fluid (CSF) donated by six patients and six healthy volunteers, who were matched for sex and age were also measured. All the baseline data were collected, and the patients underwent clinical evaluation. The correlations between CHIP levels and several clinical measurements were analyzed.Results: The serum CHIP level in the SCA3 group was (80.93 ± 28.68) ng/mL, which was significantly higher than those in the control group [(40.37 ± 18.55) ng/mL]. Similar results were observed for the CSF [(164.59 ± 42.99) ng/mL and (37.47 ± 7.85) ng/mL, respectively]. CSF CHIP levels were significantly higher than the serum CHIP levels in the SCA3 group but not in the control group. The Dunn-Bonferroni post-hoc for Kruskal-Wallis test revealed no significant difference between the serum and CSF of the patients and the control group. Multivariate linear regression showed that serum CHIP levels correlated positively with disease severity, as measured by the Scale for the Assessment and Rating of Ataxia (SARA) and the International Cooperative Ataxia Rating Scale (ICARS). Moreover, we found that serum CHIP levels were moderately correlated with age in healthy controls.Conclusion: The present study determined that CHIP levels increased significantly in the serum and CSF of patients with SCA3 and that serum CHIP levels were corelated with disease severity. Thus, CHIP is a promising biomarker for SCA3.

Previous studies have reported that PID2, which encodes a B-lectin receptor-like kinase, is a key gene in the resistance of rice to Magnaporthe oryzae strain ZB15. However, the PID2mediated downstream signalling events remain largely unknown.The U-box E3 ubiquitin ligase OsPIE3 (PID2-interacting E3) was isolated and confirmed to play key roles in PID2-mediated rice blast resistance. Yeast two-hybrid analysis showed that the armadillo repeat region of OsPIE3 is required for its interaction with PID2. Further investigation demonstrated that OsPIE3 can modify the subcellular localisation of PID2, thus promoting its nuclear recruitment from the plasma membrane for protein degradation in the ubiquitin-proteasome system. Site-directed mutagenesis of a conserved cysteine site (C230S) within the U-box domain of OsPIE3 reduces PID2 translocation and ubiquitination.Genetic analysis suggested that OsPIE3 loss-of-function mutants exhibited enhanced resistance to M. oryzae isolate ZB15, whereas mutants with overexpressed OsPIE3 exhibited reduced resistance. Furthermore, the OsPIE3/PID2-double mutant displayed a similar blast phenotype to that of the PID2 single mutant, suggesting that OsPIE3 is a negative regulator and functions along with PID2 in blast disease resistance.Our findings confirm that the E3 ubiquitin ligase OsPIE3 is necessary for PID2-mediated rice blast disease resistance regulation.

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