Abstract
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.
Highlights
Protein quality control (PCQ) involves a specialized cellular surveillance system that monitors protein integrity, identifies unfolded or damaged proteins, and either repairs or targets them for degradation
Why or how CHIP mutations drive disease is unclear. At this early stage in understanding spinocerebellar autosomal recessive 16 (SCAR16), it is imperative to establish preclinical models to help understand the pathophysiology and mechanism of the disease, as well as to use as a platform to design and test therapies. In this manuscript we identified the structural, biochemical, cellular, and in vivo repercussions of the first mutation described in SCAR16 patients using two rodent models engineered with CRISPR/Cas9 editing to mimic a disease-causing human mutation
The T246M mutation destabilizes the structure of the U-box and CHIP-T246M forms decamers and dodecamers in vitro and in cells: Asymmetric homodimerization of CHIP as well as conformational flexibility are required for CHIP ubiquitin ligase activity
Summary
Protein quality control (PCQ) involves a specialized cellular surveillance system that monitors protein integrity, identifies unfolded or damaged proteins, and either repairs or targets them for degradation. CHIP is abundantly expressed in most tissues and plays a central role in maintaining protein quality control [1]. CHIP is uniquely suited as a regulator of protein quality control due to its dual functions as both a co-chaperone protein and ubiquitin ligase enzyme. Since the discovery of CHIP in 1999 [1], numerous reports detailing CHIP’s co-chaperone and ubiquitin ligase activities in both the brain and heart have been published [4,5,6,7]. Recent reports describing surprising new roles for CHIP have emerged. These roles include autonomous chaperone activity [8,9], the regulation of cardiac metabolic homeostasis via the metabolic sensor AMPK (AMP-activated protein kinase) [9], and DNA damage repair [10]. CHIP was implicated in the pathophysiology associated with the disease spinocerebellar autosomal recessive 16 (SCAR16) [11,12], representing the first direct association between a CHIP polymorphism and a human disease
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