All cells must respond to changes in their environment including a plethora of physiologic and pathologic stresses in order to maintain homeostasis and survive. Protein homeostasis is particularly critical to cell survival and cells utilize multiple highly specialized and integrated methods of protein quality control (PQC) to ensure that proteins are appropriately folded and terminally misfolded proteins are eliminated to prevent proteotoxicity. PQC depends on an elegant collaboration between molecular chaperones and the ubiquitin-proteasome system (UPS). Disruption of PQC and subsequent proteotoxicity is an underlying molecular phenotype in disease pathologies in the brain and heart. Understanding the molecular mechanisms underlying diseases where disruption of PQC is central to disease pathology is key to our ability to intervene therapeutically. To this end, this thesis focuses on understanding the function of E3 ubiquitin ligases and how mutations in these key players in the UPS can drive disease pathology in the heart and brain. First, I describe and validate a novel method for the identification of E3 ubiquitin ligase substrates addressing a significant technological limitation in the field. Next, I describe the first discovery of human mutation in the E3 ubiquitin ligase CHIP in a form of spinocerebellar ataxia, Gordon Holmes Syndrome that has led to the establishment of a new disease designation, autosomal recessive spinocerebellar ataxia-16 (SCAR16) to describe spinocerebellar ataxia caused by homozygous or compound heterozygous mutation in CHIP. Finally, I expanded upon this discovery to define the structural and functional consequences of CHIP mutation in SCAR16 and explore the deficits associated with this mutation in a genomic context utilizing a mouse model system providing the first in vivo, disease-relevant model of partial CHIP dysfunction. Together these studies provide novel tools to further our understanding of the UPS and reveal fascinating insight into the molecular mechanisms underlying CHIP mutation in SCAR16 disease that not only may facilitate the development of therapies for this devastating disease, but also contribute to our basic understanding of the UPS and its role in disease pathogenesis to drive successful investment, innovation, preclinical investigation and clinical study design in other disease areas.