Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity.
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ABSTRACT:
SUBMITTER: Ochsenreiter T
PROVIDER: S-EPMC1679783 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1-3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.
| S-EPMC6004266 | biostudies-literature
Project description:In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.
| S-EPMC3607012 | biostudies-literature
Project description:The SAR of 4-hydroxybenzaldehyde inhibitors of the trypanosome alternative oxidase (TAO), a critical enzyme for the respiration of bloodstream forms of trypanosomes, was investigated. Replacing the aldehyde group with a methyl ester resulted in a 10-fold increase in TAO inhibition and activity against T. brucei. Remarkably, two analogues containing the 2-hydroxy-6-methyl scaffold (9e and 16e) displayed single digit nanomolar TAO inhibition, which constitute the most potent 4-alkoxybenzoic acid derivatives described to date. 9e was 50-times more potent against TAO and 10-times more active against T. brucei compared to its benzaldehyde analogue 1. The farnesyl derivative 16e was as potent a TAO inhibitor as ascofuranone with IC50 = 3.1 nM. Similar to ascofuranone derivatives, the 2-hydroxy and 6-methyl groups seemed essential for low nanomolar TAO inhibition of acid derivatives, suggesting analogous binding interactions with the TAO active site.
| S-EPMC6142067 | biostudies-literature
Project description:The editing of trypanosome mitochondrial mRNAs produces transcripts necessary for mitochondrial functions including electron transport and oxidative phosphorylation. Precursor-mRNAs are often extensively edited by specific uridine insertion or deletion that is directed by small guide RNAs (gRNAs). Recently, it has been shown that cytochrome c oxidase subunit III (COXIII) mRNAs can be alternatively edited to encode a novel mitochondrial membrane protein composed of a unique hydrophilic N-terminal sequence of unknown function and the C-terminal hydrophobic segment of COXIII. To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes. Using this data, we show that alternative editing of COXIII, ATPase subunit 6 (A6), and NADH dehydrogenase subunits 7, 8 and 9 (ND7, 8, 9) mRNAs can produce novel open reading frames (ORFs). Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified. These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms.
| S-EPMC2215773 | biostudies-literature
Project description:Human ADAM12, transcript variant 1 (later on referred to as Var-1b), present in publicly available databases contains the sequence 5'-GTAATTCTG-3' at the nucleotide positions 340-348 of the coding region, at the 3' end of exon 4. The translation product of this variant, ADAM12-Lb, includes the three amino acid motif (114)VIL(116) in the prodomain. This motif is not conserved in ADAM12 from different species and is not present in other human ADAMs. Currently, it is not clear whether a shorter variant, Var-1a, encoding the protein version without the (114)VIL(116) motif, ADAM12-La, is expressed in human. In this work, we have established that human mammary epithelial cells and breast cancer cells express both Var-1a and Var-1b transcripts. Importantly, the proteolytic processing and intracellular trafficking of the corresponding ADAM12-La and ADAM12-Lb proteins are different. While ADAM12-La is cleaved and trafficked to the cell surface in a manner similar to ADAM12 in other species, ADAM12-Lb is retained in the ER and is not proteolytically processed. Furthermore, the relative abundance of ADAM12-La and ADAM12-Lb proteins detected in several breast cancer cell lines varies significantly. We conclude that the canonical form of transmembrane ADAM12 is represented by Var-1a/ADAM12-La, rather than Var-1b/ADAM12-Lb currently featured in major sequence databases.
| S-EPMC3792144 | biostudies-literature
Project description:Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.
| S-EPMC4000096 | biostudies-literature
Project description:Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells.
| S-EPMC3378104 | biostudies-literature
Project description:Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5', in wild-typeTrypanosoma brucei We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5' UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences.
| S-EPMC4836643 | biostudies-literature
Project description:We report here the isolation of the 5' end and the promoter region of the gene for chicken cartilage link protein, and demonstrate extensive heterogeneity of the leader sequence arising from differential utilization of multiple splice sites within the 5'-most exon. The 500-base pairs (bp) exon 1 consists of solely untranslated sequence and is followed by an intron greater than 33 kilobase pairs (kb). Together, the five exons predict a gene size longer than 100 kb. Multiple transcription initiation sites were mapped 34, 46, 56, 66 and 76 bp downstream of a TATA-like motif. Sequence analysis revealed that in addition to the non-spliced variant, multiple mRNA species were generated by alternative splicing resulting in the exclusion of 92, 166, 170, 174 and 263 nucleotides (nt), respectively, from exon 1. Polymerase chain reaction confirmed the existence of various splice forms, and showed cell type- and developmental stage-specific expression for one group of them. Secondary structure predictions indicated that the leaders of the splice forms could form stable hairpin structures with different free energies of formation (up to delta G = -110 kcal/mol), suggesting translational control. The splice variant detected in the largest amount had the least stable predicted hairpin (delta G = -31.7 kcal/mol).
| S-EPMC328800 | biostudies-other
Project description:Comparative analysis of the transcriptional response, as quantified by RNA-Seq, of Mycobacterium tuberculosis (Mtb) during inhibition of the terminal cytochrome oxidases, Cytochrome bd oxidase and Cytochrome bcc:aa3. This study was designed to evaluate the efficacy if a novel small molecule inhibitor of the Cytochrome bd oxidase. Specifically, we compared the transcriptional response of Mtb during inhibition by Q203 with a Cytochrome bd deletion mutant to the transcriptional response of Mtb treated with a combination of Q203 and the purported Cytorchomre bd small molecule inhibitor (ND-011992).
2020-10-07 | GSE159080 | GEO