Abstract
The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.
Highlights
Transition from one cell cycle stage to the is achieved in eukaryotes through mechanisms that provide an all-or-none cyclin-dependent kinases (CDKs) activation
African sleeping sickness is a neglected tropical disease caused by infection with the protozoan parasite Trypanosoma brucei, which is transmitted to humans by tsetse flies (Glossina genus)
Understanding how T. brucei regulates cell cycle progression at a molecular level when alternating between the mammalian host and the insect vector could lead to better therapies
Summary
Transition from one cell cycle stage to the is achieved in eukaryotes through mechanisms that provide an all-or-none cyclin-dependent kinases (CDKs) activation The activity of these enzymes is regulated by several mechanisms, including association with regulatory subunits (cyclins), phosphorylation and dephosphorylation and interaction with CDK inhibitors (CKIs) [1, 2]. Levels of cyclins (CYC), CKIs and many other cell cycle regulators oscillate during the cell cycle as a result of periodic proteolysis, generating a unidirectional control. These proteins are targeted for degradation by polyubiquitination, i.e. the attachment of multiple copies of ubiquitin. The specificity of ubiquitin-dependent proteolysis derives from the many hundreds of E3 ubiquitin ligases that recognize particular substrates through interaction domains [4]
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