Characterisation of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei.

The mitochondrial F1Fo ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F1 moiety is relatively highly conserved in structure and composition, the Fo subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicated that the protein is both associated with the F1Fo ATP synthase, and integral to its assembly. In addition, its knockdown reduced the levels of Fo subunits, but not those of F1, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to Fo subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b

Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex

BACKGROUND: Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry. PRINCIPLE FINDINGS: The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues. CONCLUSION: Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders

Stable isotopic labeling in proteomics

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon-13, nitrogen-15, and oxygen-18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism

Protein factors involved in the biogenesis of the mitochondrial ribosome

The mammalian mitochondria contain their own genome which encodes thirteen polypeptide components of the oxidative phosphorylation (OxPhos) system, and the mitochondrial (mt-) rRNAs and tRNAs required for their translation. The maturation of the mitochondrial ribosome requires both mt-rRNAs to undergo post-transcriptional chemical modifications, folding of the rRNA and assembly of the protein components assisted by numerous biogenesis factors. The post-transcriptional modifications of the mt-rRNAs include base methylations, 2’-O-ribose methylations and pseudouridylation. However, the exact function of these modifications is unknown. Many mitoribosome biogenesis factors still remain to be identified and characterised. This work aims to broaden our understanding of two proteins involved in mitoribosome biogenesis through the study of the function of an rRNA methyltransferase and a novel biogenesis factor. Firstly, we characterised MRM1 (mitochondrial rRNA methyltransferase 1), a highly conserved 2’-O-ribose methyltransferase. We confirmed that MRM1 modifies a guanine in the peptidyl (P) transferase region of the 16S mt-rRNA that specifically interacts with the 3’ end of the tRNA at the ribosomal P-site. In bacteria, the modification is dispensable for ribosomal biogenesis and cell viability under standard conditions. However, in yeast mitochondria, Mrm1p is vital for ribosomal assembly and function. We generated knockout cells lines using programmable nuclease technology, and characterised the possible effects of MRM1 depletion on mitochondrial translation and mitoribosome biogenesis. We demonstrated that neither the enzyme nor the modification is required for human mitoribosomal assembly and translation in our experimental setup. Secondly, we identified a novel mitochondrially-targeted putative RNA endonuclease, YbeY. Using YbeY knockout cell lines, we showed that depletion of YbeY leads to loss of cell viability and OxPhos function as a consequence of a severe decrease in mitochondrial translation. Northern blotting and transcriptomic analysis using next generation RNA-Seq revealed transcript-specific changes to steady state levels. This analysis identified mt-tRNASer as a potential target of YbeY. We investigated the effect of YbeY deficiency on mitoribosomal assembly by quantitative sucrose gradient fractionation and mass spectrometry. This analysis showed that the mt-SSU is depleted in YbeY knockout cells. Further, immunoaffinity purification identified MRPS11 as a key interactor of YbeY. We propose that YbeY is a multifunctional protein that performs endonucleolytic functions in the mitochondria and also acts as a mitochondrial ribosome biogenesis factor, assisting small subunit assembly through its interaction with MRPS11.Medical Research Counci

Keeping in touch with type-III secretion system effectors : mass spectrometry-based proteomics to study effector-host protein-protein interactions

Manipulation of host cellular processes by translocated bacterial effectors is key to the success of bacterial pathogens and some symbionts. Therefore, a comprehensive understanding of effectors is of critical importance to understand infection biology. It has become increasingly clear that the identification of host protein targets contributes invaluable knowledge to the characterization of effector function during pathogenesis. Recent advances in mapping protein–protein interaction networks by means of mass spectrometry-based interactomics have enabled the identification of host targets at large-scale. In this review, we highlight mass spectrometry-driven proteomics strategies and recent advances to elucidate type-III secretion system effector–host protein–protein interactions. Furthermore, we highlight approaches for defining spatial and temporal effector–host interactions, and discuss possible avenues for studying natively delivered effectors in the context of infection. Overall, the knowledge gained when unravelling effector complexation with host factors will provide novel opportunities to control infectious disease outcomes

Examining the mechanistic regulation of starvation-induced autophagy via the identification and characterisation of novel ULK kinase substrates

Autophagy involves the formation of an endoplasmic reticulum-derived membrane termed a phagophore which expands to engulf cytoplasmic cargo before sealing to form an autophagosome. Amino acid starvation is amongst the most potent autophagic stimuli, however whilst the key signalling complexes involved in starvation-induced autophagy are known, the precise regulatory mechanisms remain poorly understood. The serine/threonine kinase ULK1 and close homolog ULK2 assume the most upstream position in the autophagic signalling cascade and play a crucial yet enigmatic role in coordinating the autophagic machinery. To further understand the mechanisms of starvation-induced autophagy, I performed a number of unbiased phosphoproteomic screens to identify ULK substrates before classifying their roles in starvation-induced autophagy. Analysis of these datasets has revealed that loss of ULK results in significant changes to the phosphoproteome and has yielded a high confidence list of potential substrates whilst also offering interesting insights into the veracity of the published ULK consensus signature. Amongst the novel phosphorylation targets are components of the retromer and AMPK complexes along with multiple components of the class III PI3K VPS34 complex. The pseudokinase p150, scaffolding component of the VPS34 complex, is phosphorylated by ULK1 in vitro and in vivo at serine 861. CRISPR-based knockout of p150 results in inhibition of autophagy and endosomal trafficking, whilst mutating the phosphorylated residue in p150 alters both omegasome establishment and autophagic flux. Furthermore, incorporation of phosphomutant p150 into the VPS34 complex modulates its lipid kinase activity in vitro. These data identify a novel ULK-dependent signalling axis and help illuminate the complexities of signal transduction in autophagy