The characterization of the tetratricopeptide repeat protein 13 in mammals

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Tetratricopeptide repeat domain protein 13 (TTC13) is a member of a large family of proteins known as tetratricopeptide repeat (TPR) proteins that has more than 5,000 members (Zeytuni 2012). One common feature of all TPR proteins is that they all contain TPR domains, which are thought to mediate protein-protein interactions and/or protein scaffolding. Interestingly, these TPR proteins are highly conserved between species. For example, sequence similarity between SUPPRESSOR of rps4 -RLD (SRFR1) and TTC13 was not only restricted to the TPR domain but also observed throughout the full length of both proteins. Extensive evidence suggested that SRFR1 was involved in pathogen resistance in plants by regulating resistance proteins in Arabidopsis thaliana (Kwon 2009). However, the mechanism by which SRFR1 regulated resistance proteins still remains unresolved. In contrast, nothing is known about the mammalian ortholog of SRFR1, TTC13. The goal of my project was to characterize TTC13 and to, ultimately, determine if TTC13 serves a similar role in mammals as SRFR1 in plants. Sequence analysis revealed that SRFR1 had 26% identity and 46% similarity at the amino acid level with TTC13. To begin addressing the role of TTC13 in mammals, I analyzed the expression of TTC13 mRNA and protein in mouse tissues. These analyses indicated that TTC13 mRNA was ubiquitously expressed in all examined mouse tissues. However, TTC13 protein expression varied in each tissue. For example, TTC13 protein was highly expressed in both kidney and liver, whereas TTC13 protein expression was not detectable in other tissues that were positive for TTC13 mRNA. Moreover, moderate protein expression was detected in the lung, heart and immune tissues (e.g. thymus, spleen, lymph nodes, T and B cells). To determine the cellular distribution of TTC13, I performed immunoflouresence and subcellular fractionation experiments. pFlag-TTC13 was localized to the cytoplasm in COS-1 cells by immunofluorescence. Cytoplasmic localization was confirmed by immunoblotting cytoplasmic and nuclear fractions for TTC13. Taken together, current evidence suggested that TTC13 was a cytoplasmic protein that was highly expressed in kidney and liver. Interestingly, TTC13 was also expressed in mammalian immune tissues in low to moderate levels, suggesting that TTC13 may have a role in the mammalian immune system. I conducted cell viability and cell cycle experiments in mammalian cell lines to investigate if TTC13 functions similarly to other TPR proteins.