The first part in my thesis research is on the regulation of ANO1 (TMEM16A). ANO1 protein is a calcium-activated chloride channel that plays critical roles in sensory transduction, epithelial secretion, smooth muscle contraction, and cell proliferation/migration. ANO1 mRNA and protein levels are altered under various physiological and pathological conditions; however, the molecular mechanisms through which ANO1 protein levels are altered under these conditions are unclear. The ubiquitin system potently regulates the expression of numerous ion channels, particularly their insertion into and retrieval from the cell surface, but almost nothing is known about ANO1 ubiquitination. By using the yeast-two hybrid assays, we identified TRIM23 E3 ligase as a binding partner of ANO1. The physical...[ Read more ]

The first part in my thesis research is on the regulation of ANO1 (TMEM16A). ANO1 protein is a calcium-activated chloride channel that plays critical roles in sensory transduction, epithelial secretion, smooth muscle contraction, and cell proliferation/migration. ANO1 mRNA and protein levels are altered under various physiological and pathological conditions; however, the molecular mechanisms through which ANO1 protein levels are altered under these conditions are unclear. The ubiquitin system potently regulates the expression of numerous ion channels, particularly their insertion into and retrieval from the cell surface, but almost nothing is known about ANO1 ubiquitination. By using the yeast-two hybrid assays, we identified TRIM23 E3 ligase as a binding partner of ANO1. The physical interaction between TRIM23 and ANO1, specifically the carboxyl terminus of ANO1, was further confirmed through GST-pull down and co-immunoprecipitation assays. More importantly, ANO1 is ubiquitinated and stabilized by TRIM23 both in-vivo and in-vitro. Knocking out TRIM23 in breast cancer ZR-75-1 cell line using CRISPR/Cas9 techniques robustly decreased the protein expression and reduced the ubiquitination of endogenous ANO1. In addition, EGF enhanced the protein expression of ANO1 with a TRIM23-dependent post translational mechanism. On the other hand, TRIM21 was proposed to bind to ANO1 based on a previous interactome study of ANO1. We confirmed the TRIM21-ANO1 interaction by using GST-pull down and co-immunoprecipitation assays. In contrast to TRIM23, TRIM21 downregulated the protein expression of ANO1 both in cells and in animals. Knocking out TRIM21 increased saliva secretion triggered by muscarine receptor agonist in mice. Our findings suggest that ANO1 is regulated by TRIM23 and TRIM21 E3 ligases in an antagonizing manner. This previously unrecognized regulatory mechanism may play a role in various physiological processes.

The second part of my thesis research is on the regulation of TMC1. TMC1 was proposed to be the most promising candidate for the mechanoelectrical transduction channel in the auditory hair cells because it localizes to the tips of stereocilia in the hair cells and is indispensable for auditory function. However, studying the intrinsic properties of TMC1 is impeded because TMC1 is restricted to the endoplasmic reticulum in heterologous systems. We identified the COPII adaptor TMED3 as a binding patterner of TMC1 through yeast-two hybrid assays. The physical and functional interactions between these two proteins was confirmed in vitro. Strikingly, TMED3 promoted the ER-to-Golgi translocation of TMC1. However, knocking out TMED3 did not cause deafness phenotype in mice. We reasoned that another protein may compensate TMED3 in the auditory hair cells. TMED7 was found to be highly homologous to TMED3, and we further validated the interaction of TMED7 and TMC1 in vitro. Unexpectedly, TMED7 seemingly localizes to the tips of stereocilia in the outer hair cells based on immunohistochemical assays. In addition to the post-translational regulatory mechanisms, we also examined the alternative splicing of TMC1. Tmc1 is alternatively spliced to produce protein isoforms in the auditory hair cells. Our work provides interesting insights in trafficking and alternative splicing of TMC1; these findings may pave the way for us to understand the plasma membrane localization of TMC1, especially in the heterologous systems.