Biochemical and Genetic Analysis of the E3 Ligase CHIP (STUB1): Insights Into CHIP’s Role in the Nervous System and in Spinocerebellar Ataxia Type 48 (SCA48)

Abstract

A key player in protein quality control pathways is the E3 ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP). CHIP is encoded by the STUB1 gene and is well studied for its ability to bind cellular chaperones and ubiquitinate damaged or misfolded proteins for degradation. In particular, there is a large body of literature that has investigated how CHIP interacts with proteins known to cause neurodegenerative diseases. Interestingly, mutations in CHIP itself have been found to cause neurodegenerative disease – autosomal recessive spinocerebellar ataxia type 16 (SCAR16) and spinocerebellar ataxia type 48 (SCA48). The mutations that cause these diseases span the functional domains of CHIP. While a lot of studies have now investigated the effect that disease causing mutations have on CHIP function, the full molecular pathogenesis of these two diseases have not yet been elucidated. Here, I have characterized the effects of a novel de novo missense mutation in CHIP’s TPR domain. This mutation is a missense mutation, where a single nucleotide change converts the 52nd amino acid from an alanine to a glycine. The mutation was identified in a patient with no family history of ataxia. Using a wide range of biophysical, biochemical, and cellular assays, I found that this mutation impairs CHIP’s ability to bind to chaperones but does not affect its ubiquitin ligase activity. I also found that the overexpression of CHIPA52G reduced cellular fitness of HEK293 cells in response to certain stressors, suggesting this mutation affects CHIP’s role in the cellular stress response. To investigate if this novel mutation does indeed induce neurodegeneration, I used a newly generated Caenorhabditis elegans animal model of SCA48. In this model, we found that expression of human CHIPA52G in neurons did induce neurodegeneration. These findings add to our understanding of the kinds of mutations in CHIP that can disrupt its normal function and lead to disease. Additionally, the newly established C. elegans model of SCA48 can be used to provide more information about the cellular mechanism of disease and to test future drug therapies for this disease. In addition to characterizing a novel mutation in CHIP, I sought to further understand the role of CHIP in cellular and behavioral processes. While there are numerous studies that have investigated CHIP’s role in the cell, many of them have focused on its function as a ubiquitin ligase that targets proteins for degradation. However, the specific pathways in which CHIP function is most important, especially in the context of neurodegenerative disease, remain unclear. To address this, I utilized two available Drosophila melanogaster lines. The STUB1 (CHIP) CRIMIC line was used to characterize the expression pattern of CHIP in the nervous system. I observed CHIP expression in neurons, most likely motor neurons, and muscles. Interestingly, I found varying levels of CHIP expression in a subset of glial cells. Using a STUB1 null Drosophila line, I investigated the effect of CHIP in cellular and behavioral processes. I found that CHIP is important in the embryo and larval stages of the fly life cycle. In adult flies, the absence of CHIP lead to a decrease in survival and in climbing ability. Interestingly, I found that although absence of CHIP leads to a decreased percentage of larvae pupating, it did not affect crawling ability. Finally, I found that STUB1 null larvae display a morphological defect in the neuromuscular junction boutons. This defect may affect function of the boutons, as I observed a decrease in synapsin and GluRII staining. Together, this work identifies cells in the nervous system where CHIP may be most important, reveals an important role for CHIP in Drosophila embryo and larval life stages, and suggests a possible role for CHIP in synaptic transmission.