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.

Monogenetic disorders that cause cerebellar ataxia are characterized by defects in gait and atrophy of the cerebellum; however, patients often suffer from a spectrum of disease, complicating treatment options. Spinocerebellar ataxia autosomal recessive 16 (SCAR16) is caused by coding mutations in STUB1, a gene that encodes the multifunctional enzyme CHIP (C terminus of HSC70-interacting protein). The disease spectrum of SCAR16 includes a varying age of disease onset, cognitive dysfunction, increased tendon reflex, and hypogonadism. Although SCAR16 mutations span the multiple functional domains of CHIP, it is unclear whether the location of the mutation and the change in the biochemical properties of CHIP contributes to the clinical spectrum of SCAR16. In this study, we examined relationships between the clinical phenotypes of SCAR16 patients and the changes in biophysical, biochemical, and functional properties of the corresponding mutated protein. We found that the severity of ataxia did not correlate with age of onset; however, cognitive dysfunction, increased tendon reflex, and ancestry were able to predict 54% of the variation in ataxia severity. We further identified domain-specific relationships between biochemical changes in CHIP and clinical phenotypes and specific biochemical activities that associate selectively with either increased tendon reflex or cognitive dysfunction, suggesting that specific changes to CHIP–HSC70 dynamics contribute to the clinical spectrum of SCAR16. Finally, linear models of SCAR16 as a function of the biochemical properties of CHIP support the concept that further inhibiting mutant CHIP activity lessens disease severity and may be useful in the design of patient-specific targeted approaches to treat SCAR16.

Monogenetic disorders that cause cerebellar ataxia are characterized by defects in gait and atrophy of the cerebellum; however, patients often suffer from a spectrum of disease, complicating treatment options. Spinocerebellar ataxia autosomal recessive 16 (SCAR16) is caused by coding mutations in STUB1, a gene that encodes the multi-functional enzyme CHIP (C-terminus of HSC70-interacting protein). The spectrum of disease found in SCAR16 patients includes a wide range in the age of disease onset, cognitive dysfunction, increased tendon reflex, and hypogonadism. Although SCAR16 mutations span the multiple functional domains of CHIP, it is unclear if the location of the mutation contributes to the clinical spectrum of SCAR16 or with changes in the biochemical properties of CHIP. In this study, we examined the associations and relationships between the clinical phenotypes of SCAR16 patients and how they relate to changes in the biophysical, biochemical, and functional properties of the corresponding mutated protein. We found that the severity of ataxia did not correlate with age of onset; however, cognitive dysfunction, increased tendon reflex, and ancestry were able to predict 54% of the variation in ataxia severity. We further identified domain-specific relationships between biochemical changes in CHIP and clinical phenotypes, and specific biochemical activities that associate selectively to either increased tendon reflex or cognitive dysfunction, suggesting that specific changes to CHIP-HSC70 dynamics contributes to the clinical spectrum of SCAR16. Finally, linear models of SCAR16 as a function of the biochemical properties of CHIP support the concept that further inhibiting mutant CHIP activity lessens disease severity and may be useful in the design of patient-specific targeted approaches to treat SCAR16. ataxia | aging | ubiquitin | protein quality control | modeling disease 1 These authors contributed equally to this work 2

One out of every four deaths in the United States can be attributed to heart disease. Atherosclerosis is a prevalent form of cardiovascular disease that describes the narrowing of arteries, leading to the restriction of blood flow. Atherosclerosis is caused in part by the accumulation of lipid‐laden macrophages (foam cells) in the walls of arteries. Determining mechanisms that influence lipid metabolism in macrophages may uncover new approaches to modify the disease process and decrease the disease burden. Recently, our lab identified a genetic link between the expression of the chemokine CXCL5 and atherosclerosis in patients that is consistent with a cardioprotective role for CXCL5. One effect of CXCL5 appears to be in the regulation of reverse cholesterol transporters. We found that exposure to CXCL5 decreases foam cell forming macrophages, suggesting CXCL5 may directly impact lipid metabolism. What is not known is how CXCL5 modifies the pathophysiological response to conditions that contribute to atherosclerosis, such as a high fat diet (HFD). We hypothesized that CXCL5 mediates cardioprotection by altering the physiological response to a HFD, such that, lower levels of CXCL5 will lead to altered lipid handling and predisposition to more severe atherosclerosis. We challenged wild‐type (WT) and CXCL5−/− mice with either a HFD (0.2% cholesterol, 42% calories from fat, N = 17 per genotype), or normal chow (NC, 0% cholesterol, 16% calories from fat, N = 15 per genotype) for 16 w. To determine the effect of the diet on circulating lipid levels, we performed blood lipid analysis after the feeding regimen. Interestingly, HFD did not increase cholesterol levels to the same extent in CXCL5−/− mice as compared to WT mice. Specifically, we found that WT mice had a robust, diet‐dependent increase in both high‐density lipoprotein and low‐density lipoprotein levels (LDL: 59.6 mg/dl vs 11.3 mg/dl and HDL: 118.7 mg/dl vs 68.1 mg/dl in HFD vs NC, respectively), whereas in CXCL5−/− mice, this response was diminished (LDL: 20.6 mg/dl vs 8.8 mg/dl and HDL: 82.6 mg/dl vs 56.8 mg/dl in HFD vs NC, respectively). These results suggest that the genetic depletion of CXCL5 alters lipid handling in vivo. Additionally, white blood cell analysis identified that WT mice responded to the HFD with an increase in the number of lymphocytes (10.3e3 cells/μl vs 5.2e3 cells/μl in HFD WT vs NC WT, p = 0.0224) and basophils (20 cells/μl vs 4.0 cells/μl, p = 0.0038), an effect that was attenuated in KO mice in response to the HFD (p > 0.05). These findings suggest that CXCL5 may modify a specific component in the chronic inflammatory response to the HFD. Unexpectedly, HFD led to a modest decrease in cardiac function in KO mice after 16 w of the HFD compared to WT mice (74% vs 82% ejection fraction in CXCL5−/− vs WT mice, respectively, p = 0.0058). These preliminary findings suggest that CXCL5 plays a multifaceted role in the cardiovascular system’s response to an atherogenic diet. Currently, studies are underway using athero‐prone mice to study the e...