Functional characterization of the p13 protein of HTLV-1

HTLV-1 is the causative agent of adult T-cell leukemia-lymphoma (ATLL), an aggressive neoplasm of mature CD4+ T cells refractory to current therapies, and of HAM/TSP, a neurodegenerative disease. HTLV-1 is a complex retrovirus whose genome encodes several regulatory and accessory proteins (Rex, Tax, HBZ, p12, p30, p13) in addition to the structural proteins (gag, pol, env) common to all retroviruses. Previous studies demonstrated that HTLV-1 p13, an 87-amino acid protein, is targeted to the inner membrane of mitochondria and induces mitochondrial fragmentation. In vitro studies carried out using the full-length synthetic protein and isolated mitochondria showed that p13 induces a potential-dependent potassium (K+) influx into the mitochondrial matrix, accompanied by a dose-dependent depolarization, and reduces the threshold for permeability transition pore (PTP) opening. This effect on K+ permeability is mediated by an amphipatic α-helical domain (amino acids 21-30), in which four positively charged arginines play a key role. p13 slows down cell proliferation, promotes apoptosis triggered by ceramide and Fas L, and interferes with tumor growth in experimental models. The studies described in the present thesis were aimed at testing the effects of p13 on mitochondria in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we demonstrated that p13 induces dose-dependent mitochondrial depolarization of HeLa cells. The p13RQ mutant, which is inactive on K+ permeability, had no effect on mitochondrial membrane potential (∆ψ). Similar results were obtained with Jurkat T-cells. As ∆ψ is a key factor controlling mitochondrial calcium (Ca2+) uptake, we analyzed the effect of p13 on Ca2+ homeostasis. To this end, we employed organelle-targeted aequorin and showed that p13 specifically reduced mitochondrial Ca2+ uptake, upon histamine stimulation, while it does not alter Ca2+ concentration in the cytoplasm or the endoplasmic reticulum (ER). This effect suggests that p13 might control key processes regulated through Ca2+ signalling, such as T-cell activation and death. The observation that p13's effect on ∆ψ was heterogeneous at intermediate levels of expression suggested the existence of mechanisms regulating p13 function. In the present study, we investigated the possible role of phosphorylation in the regulation of p13 function. In particular, we focused on serine 15 (S15), a putative protein kinase C (PKC) phosphorylation site located in the proximity of the α-helical domain. In vitro phosphorylation assays on p13[9-22] and p13S15A[9-22] synthetic peptides confirmed the phosphorylation of S15 by purified PKC and by cytosolic and mitochondrial lysates from Jurkat T-cells as a source of kinases. We also confirmed the in vitro phosphorylation of the full-length synthetic p13 by Jurkat T-cell lysates. Preliminary results obtained by 2D-electrophoresis analysis of Jurkat T-cells transiently transfected with p13 demonstrated that the protein is detected in two main forms differing in their pI. We then analyzed the functional effects of S15 phosphorylation by producing the phosphoablative (p13S15A) and phosphomimetic (p13S15D) mutants and studying their properties in HeLa cells. Results demonstrated that the presence of a negative charge on residue 15 enhances the effect of p13 on the induction of mitochondrial fragmentation. Interestingly, using TMRM, we found that the phosphomimetic mutant induces mitochondrial depolarization at much lower level of expression, compared to p13 wild-type. Taken together, these results suggest that phosphorylation on S15 might influence the biological properties of p13, and open the door to future studies of this and other cellular mechanisms regulating p13 function.