The essential cysteines in the CIPC motif of the thioredoxin-like Trypanosoma brucei MICOS subunit TbMic20 do not form an intramolecular disulfide bridge in vivo.

The mitochondrial protein import machinery of trypanosomatids is highly divergent from that of the well-studied models such as baker's yeast. A notable example is that the central catalyst of the mitochondrial intermembrane space import and assembly pathway (MIA), named Mia40, is missing in trypanosomatids. Mia40 works in a two-step process. First it recognizes by direct binding reduced MIA substrate proteins and then catalyzes their oxidative folding to produce intramolecular disulfide bridges. It was recently proposed that a thioredoxin-like subunit of the trypanosomal mitochondrial contact site and cristae organizing system (MICOS) called TbMic20 may be the Mia40 replacement. Our study performed on procyclic stage of the parasite revealed that each of the two cysteines in TbMic20's active site is essential for the stability of MIA substrate proteins although they do not form a disulfide bridge in vivo. The two cysteines of Mia40's active site form an intramolecular disulfide bridge at steady state, which is a prerequisite for its oxidative folding of MIA substrates. Thus, we conclude that TbMic20 is unlikely to represent a bona fide Mia40 replacement and plays a still unresolved role in the stability and/or import of MIA substrates in trypanosomatids. Despite this, the effect of TbMic20 depletion and mutation indicates that the trypanosomal MICOS complex still plays a vital role in the maturation and/or stability of proteins imported by the MIA pathway.

Futile import of tRNAs and proteins into the mitochondrion of Trypanosoma brucei evansi.

Trypanosoma brucei brucei has two distinct developmental stages, the procyclic stage in the insect and the bloodstream stage in the mammalian host. The significance of each developmental stage is punctuated by specific changes in metabolism. In the insect, T. b. brucei is strictly dependent on mitochondrial function and thus respiration to generate the bulk of its ATP, whereas in the mammalian host it relies heavily on glycolysis. These observations have raised questions about the importance of mitochondrial function in the bloodstream stage. Peculiarly, akinetoplastic strains of Trypanosoma brucei evansi that lack mitochondrial DNA do exist in the wild and are developmentally locked in the glycolysis-dependent bloodstream stage. Using RNAi we show that two mitochondrion-imported proteins, mitochondrial RNA polymerase and guide RNA associated protein 1, are still imported into the nucleic acids-lacking organelle of T. b. evansi, making the need for these proteins futile. We also show that, like in the T. b. brucei procyclic stage, the mitochondria of both bloodstream stage of T. b. brucei and T. b. evansi import various tRNAs, including those that undergo thiolation. However, we were unable to detect mitochondrial thiolation in the akinetoplastic organelle. Taken together, these data suggest a lack of connection between nuclear and mitochondrial communication in strains of T. b. evansi that lost mitochondrial genome and that do not required an insect vector for survival.

The assembly of F(1)F(O)-ATP synthase is disrupted upon interference of RNA editing in Trypanosoma brucei.

Throughout eukaryotes, the gene encoding subunit 6 (ATP6) of the F(1)F(O)-ATP synthase (complex V) is maintained in mitochondrial (mt) genomes, presumably because of its high hydrophobicity due to its incorporation into the membrane-bound F(O) moiety. In Trypanosoma species, a mt transcript that undergoes extensive processing by RNA editing has a very low sequence similarity to ATP6 from other organisms. The notion that the putative ATP6 subunit is assembled into the F(O) sub-complex is ostensibly challenged by the existence of naturally occurring dyskinetoplastic (Dk) and akinetoplastid (Ak) trypanosomes, which are viable despite lacking the mtDNA required for its expression. Taking advantage of the different phenotypes between RNA interference knock-down cell lines in which the expression of proteins involved in mtRNA metabolism and editing can be silenced, we provide support for the view that ATP6 is encoded in the mt genome of Trypanosoma species and that it is incorporated into complex V. The reduction of the F(1)F(O) oligomer of complex V coincides with the accumulation of the F(1) moiety in ATP6-lacking cells, which also appear to lack the F(O) ATP9 multimeric ring. The oligomycin sensitivity of ATPase activity of complex V in ATP6-lacking cells is reduced, reflecting the insensitivity of the Dk and Ak cells to this drug. In addition, the F(1) moiety of complex V appears to exist as a dimer in steady state conditions and contains the ATP4 subunit traditionally assigned to the F(O) sub-complex.

Generalised lymphadenopathy as the first manifestation of lupus nephritis.

Fever with generalised lymphadenopathy is a common presentation in clinical practice. A degree of lymphadenopathy is frequently a characteristic of established systemic lupus erythematosus (SLE), but it is rarely the primary presenting feature. A 25-year-old man presented with night sweats, weight loss and generalised lymphadenopathy. A chest computed tomography scan confirmed the presence of mediastinal, hilar and axillary lymphadenopathy, with bilateral pleural effusions. The double stranded DNA antibody (anti-dsDNA) was absent. Subsequently, there was mild renal impairment and a renal biopsy showed lupus nephritis. Anti-dsDNA was positive using an alternative assay. Treatment with prednisolone and mycophenolate mofetil led to considerable clinical improvement. Extensive lymphadenopathy as the first clinical manifestation of SLE is rare and this case also illustrates the variable results obtained from different anti-dsDNA antibody assays.