Identification of a Bacterial-Like HslVU Protease in the Mitochondria of Trypanosoma brucei and Its Role in Mitochondrial DNA Replication

Open Access
Publisher Website
Google Scholar
Abstract
ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms. ATP-dependent protein-hydrolyzing enzyme complexes are present in all living organisms, including the 26S proteasome in eukaryotes and the HslVU complex in bacteria. A simultaneous presence of both complexes in an organism was originally deemed unlikely until some HslVU homologs were found in certain ancient eukaryotes, though their potential function in these organisms remains unclear. We characterized an HslVU complex in Trypanosoma brucei, a protozoan parasite that causes human sleeping sickness in Africa. The complex is an active enzyme localized to the mitochondria of the parasite and closely associated with the mitochondrial DNA complex, which consists of several thousand small circular DNAs and a few dozen mitochondrial genomic DNAs. Depletion of this HslVU from the parasite resulted in a continuous synthesis of the small circular DNA, which led to aberrant segregation and incessant growth of the mitochondrial DNA complex to an enormous size that eventually blocks cell division. This novel HslVU function, which has not been observed in other organisms previously, could be a potential target for anti-sleeping sickness chemotherapy.