Leishmania donovani: proteasome-mediated down-regulation of methionine adenosyltransferase

Abstract

Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and 'mock-transfected' leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.

Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays

“…Proteasome targeting of the NanoLuc-PEST enzyme was assessed in the presence of the proteasome inhibitor MG-132 ( S4 Fig ). [ 43 ] Addition of 5 μM MG-132 produced a statistically significant increase in bioluminescence relative to the DMSO control ( S4A Fig ; p = 0.0272 and 0.0007 in axenic amastigotes and promastigotes respectively). In the presence of both MG-132 and cycloheximide, bioluminescence values did decrease, but were still significantly higher than in the presence of cycloheximide alone ( S4B Fig ; p = 0.0219 and 0.0010 in axenic amastigotes and promastigotes respectively).…”

“…Proteosome assays were performed using the method above with the addition of 5 μM MG-132 (Sigma) for 6 hours. [ 43 ] Statistical significance was determined using a paired, two-tailed T-test (GraphPad Prism 6.0).…”

Altered expression of an RBP‐associated arginine methyltransferase 7 in Leishmania major affects parasite infection

“…AdoMet is the methyl donor for PRMTs during methylation, resulting in AdoHcy formation (S‐adenosyl homocysteine) (Bedford and Clarke, ). Some groups have reported that the intracellular AdoMet/AdoHcy ratio in Leishmania are under strict control to avoid cell death (Perez‐Pertejo et al ., ). We have obtained proteomic data that suggest the differential expression of proteins involved in AdoMet synthesis in the Lmj PRMT7 transfectants (unpublished results).…”

Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1

“…A lower concentration (5 µM) of MG132, which was reported previously (Perez-Pertejo et al, 2011), also elicited a similar change in cell shape. In addition, MG132 slowed down the growth of the promastigotes.…”