The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in Arabidopsis. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. AtRDUF1 expression is upregulated by salt treatment. Overexpression of AtRDUF1 in Arabidopsis results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of AtRDUF1 and its close homolog AtRDUF2 (atrduf1atrduf2) was hypersensitive to salt treatment. The expression levels of the stress-response genes RD29B, RD22, and KIN1 are more sensitive to salt treatment in AtRDUF1 overexpression plants. In summary, our data show that AtRDUF1 positively regulates responses to salt stress in Arabidopsis.     

Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.     

A Trk/HKT-Type K+ Transporter from Trypanosoma brucei

The molecular mechanisms of K⁺ homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K⁺ from their hosts’ body fluids. The T. brucei genome contains two putative K⁺ channel genes, yet the trypanosomes are insensitive to K⁺ antagonists and K⁺ channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K⁺ concentrations. However, the trypanosomes are extremely sensitive to K⁺ ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na⁺-independent K⁺ transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K⁺ transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.Potassium (K⁺) is the most abundant cation in the cytosol of any cell and hence an essential macronutrient for life on earth. Concentrative K⁺ uptake across the plasma membrane is energized directly by ATPases and indirectly by the negative membrane potential or by coupling, via symport or antiport, to other transport processes such as H⁺ flux. The ancestry of K⁺ transporters renders them ideal subjects for phylogenetic comparisons. Indeed, the different kinds of known K⁺ transporters—pumps, channels, permeases, symporters, and antiporters—are all found in bacteria (43). Eukaryotes do not appear to have invented further mechanisms of K⁺ transport; on the contrary, some families of K⁺ transporters were lost over the course of eukaryote evolution, particularly among the metazoa (53).The Trk/HKT superfamily (TC transporter classification 2.A.38 [43]) consists of bacterial TrkH and KtrB, plant HKT, and fungal Trk transporters (15). These proteins share a topology with 8 transmembrane (TM) domains and, sandwiched between odd- and even-numbered TM domains, 4 shorter hydrophobic helices that resemble the P-loops of K⁺ channels (14, 27, 55). In the K⁺ channel, these pore-forming loops end in the filter residues glycine-tyrosine-glycine, which coordinate K⁺ by means of their backbones’ carbonyl oxygens (13). The P-loop-like helices of Trk/HKT transporters end in a single conserved glycine (48), and these glycines have been shown to determine K⁺ selectivity over Na⁺ of the transporters (34, 49). Thus, a Trk/HKT monomer with 8 TM domains and 4 P-loops is thought to have a similar pore architecture to a K⁺ channel tetramer with two TM domains and one P-loop per subunit. The Trk/HKT transporters are important for cellular K⁺ acquisition in microorganisms, since trk null mutant yeast or bacteria exhibit growth phenotypes on media containing low K⁺ concentrations (20, 46). The roles of the Trk/HKT transporters in plants are more diverse, including Na⁺ distribution (10, 33, 47), osmoregulation (32), and salt tolerance (39). So far, no HKT/Trk transporter has been described from the metazoa or protista.Trypanosoma brucei subsp. parasites comprise the causative agents of human and livestock trypanosomosis: sleeping sickness and nagana, respectively. The distribution of the parasites is restricted by that of their vector, the blood-sucking tsetse fly (Glossina spp.), to the so-called tsetse belt comprising 36 countries between the Sahara desert and the Kalahari (3, 8). African trypanosomes proliferate extracellularly in the blood, evading the mammalian immune response by antigenic variation. Untreated sleeping sickness is fatal. There is an urgent need for new and better drugs since the current ones, the arsenical melarsoprol in particular, suffer from severe side effects (31). In the mammalian bloodstream, the parasites encounter a rich and steady supply of nutrients, readily imported by specific permeases or endocytosed via receptors (7). Research on trypanosomal nutrient uptake has so far concentrated on transporters of organic substrates: nucleobases, nucleosides, sugars, and amino acids (4, 12, 26, 30, 35, 56). Little is known about how the parasites import inorganic nutrients. The malaria parasite Plasmodium falciparum possesses two putative K⁺ channel subunits with 6 TM domains and one P-loop (19, 52). Disruption of an orthologous gene in Plasmodium berghei strongly impaired the development of the malaria parasites in the mosquito (18). However, these putative channels have not yet been proven to be permeable to K⁺. The T. brucei genome (6) is annotated to contain two putative K⁺ channels; in addition, a putative ATPase has been identified resembling fungal Na⁺/K⁺ efflux ATPases (5, 45). None of these has been addressed experimentally. Here we present the identification and characterization of TbHKT1 (Tb10.70.2940), a Trk/HKT-type K⁺ transporter from Trypanosoma brucei and representative of a new clade of Trk/HKT genes from kinetoplastid parasites.     

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.     

Differential functions of two editosome exoUases in Trypanosoma brucei

Mitochondrial RNAs in trypanosomes are edited by the insertion and deletion of uridine (U) nucleotides to form translatable mRNAs. Editing is catalyzed by three distinct editosomes that contain two related U-specific exonucleases (exoUases), KREX1 and KREX2, with the former present exclusively in KREN1 editosomes and the latter present in all editosomes. We show here that repression of KREX1 expression leads to a concomitant reduction of KREN1 in ∼20S editosomes, whereas KREX2 repression results in reductions of KREPA2 and KREL1 in ∼20S editosomes. Knockdown of KREX1 results in reduced cell viability, reduction of some edited RNA in vivo, and a significant reduction in deletion but not insertion endonuclease activity in vitro. In contrast, KREX2 knockdown does not affect cell growth or editing in vivo but results in modest reductions of both insertion and deletion endonuclease activities and a significant reduction of U removal in vitro. Simultaneous knockdown of both proteins leads to a more severe inhibition of cell growth and editing in vivo and an additive effect on endonuclease cleavage in vitro. Taken together, these results indicate that both KREX1 and KREX2 are important for retention of other proteins in editosomes, and suggest that the reduction in cell viability upon KREX1 knockdown is likely a consequence of KREN1 loss. Furthermore, although KREX2 appears dispensable for cell growth, the increased inhibition of editing and parasite viability upon knockdown of both KREX1 and KREX2 together suggests that both proteins have roles in editing.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Role of Centrins 2 and 3 in Organelle Segregation and Cytokinesis in Trypanosoma brucei

Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2) and T. brucei 3 (TbCen3) in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1–3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.     

Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact.     

TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.     

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.