TY - GEN

T1 - Investigating the role of Deleted in Primary Ciliary Dyskinesia (DPCD) protein in Trypanosoma brucei

AU - Ablett, Emily

PY - 2023

Y1 - 2023

N2 - Primary Ciliary Dyskinesia (PCD) is the most common ciliary syndrome in humans, associated with the reduction or loss of function of motile cilia due to inherited mutations. This results in life-altering symptoms spanning multiple organ systems, including airway infections and infertility. Since its discovery in 2004, Deleted in Primary Ciliary Dyskinesia (DPCD) protein has become a novel gene candidate for PCD, with a predicted role in the formation of cilia by ciliogenesis. However, the precise role of this protein in ciliogenesis remains obscure. To provide further insight into the role of this enigmatic protein, the Trypanosoma brucei homologue of DPCD was investigated. Bioinformatics interrogation revealed that DPCD is strongly conserved across ciliated eukaryotes but that a homologue of DPCD is only found in eukaryotes with motile cilia supporting a proposed role in ciliogenesis. However, endogenous tagging of the TbDPCD protein in procyclic T. brucei cells localised the protein to the cell cytoplasm, while RNAi induced ablation of TbDPCD produced no observable effect on cell growth, morphology, motility, or cell cycle progression. This suggests that TbDPCD is not essential for ciliogenesis in T. brucei or potentially has a redundant function. The in-silico interrogation of DPCD protein structure/function using Phyre2 and Alphafold predicted that DPCD has structural similarity to proteins that have chaperone or co-chaperone activity, including small heat-shock proteins. Consequently, the TbDPCD protein was overexpressed and purified from Escherichia coli to determine whether TbDPCD demonstrated chaperone activity under heat stress conditions using a thermal aggregation assay. These experiments could not demonstrate that TbDPCD possesses chaperone activity under heat stress, however, the results indicated that TbDPCD is not stable at elevated temperatures. Overall, the in-silico analysis gives a strong indication of the role of DPCD as a chaperone, however in vivo and biochemical studies did not provide evidence to support this.

AB - Primary Ciliary Dyskinesia (PCD) is the most common ciliary syndrome in humans, associated with the reduction or loss of function of motile cilia due to inherited mutations. This results in life-altering symptoms spanning multiple organ systems, including airway infections and infertility. Since its discovery in 2004, Deleted in Primary Ciliary Dyskinesia (DPCD) protein has become a novel gene candidate for PCD, with a predicted role in the formation of cilia by ciliogenesis. However, the precise role of this protein in ciliogenesis remains obscure. To provide further insight into the role of this enigmatic protein, the Trypanosoma brucei homologue of DPCD was investigated. Bioinformatics interrogation revealed that DPCD is strongly conserved across ciliated eukaryotes but that a homologue of DPCD is only found in eukaryotes with motile cilia supporting a proposed role in ciliogenesis. However, endogenous tagging of the TbDPCD protein in procyclic T. brucei cells localised the protein to the cell cytoplasm, while RNAi induced ablation of TbDPCD produced no observable effect on cell growth, morphology, motility, or cell cycle progression. This suggests that TbDPCD is not essential for ciliogenesis in T. brucei or potentially has a redundant function. The in-silico interrogation of DPCD protein structure/function using Phyre2 and Alphafold predicted that DPCD has structural similarity to proteins that have chaperone or co-chaperone activity, including small heat-shock proteins. Consequently, the TbDPCD protein was overexpressed and purified from Escherichia coli to determine whether TbDPCD demonstrated chaperone activity under heat stress conditions using a thermal aggregation assay. These experiments could not demonstrate that TbDPCD possesses chaperone activity under heat stress, however, the results indicated that TbDPCD is not stable at elevated temperatures. Overall, the in-silico analysis gives a strong indication of the role of DPCD as a chaperone, however in vivo and biochemical studies did not provide evidence to support this.

U2 - 10.17635/lancaster/thesis/1912

DO - 10.17635/lancaster/thesis/1912

M3 - Master's Thesis

PB - Lancaster University

ER -