A cDNA encoding a trypsin-like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin-like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.
The F1F0-ATP synthase translates a proton flux across the inner mitochondrial membrane into a mechanical rotation, driving anhydride bond formation in the catalytic portion. The complex's membrane-embedded motor forms a proteinaceous channel at the interface between Atp9 ring and Atp6. To prevent unrestricted proton flow dissipating the H+-gradient, channel formation is a critical and tightly controlled step during ATP synthase assembly. Here we show that the INA complex (INAC) acts at this decisive step promoting Atp9-ring association with Atp6. INAC binds to newly synthesized mitochondrial-encoded Atp6 and Atp8 in complex with maturation factors. INAC association is retained until the F1-portion is built on Atp6/8 and loss of INAC causes accumulation of the free F1. An independent complex is formed between INAC and the Atp9 ring. We conclude that INAC maintains assembly intermediates of the F1 F0-ATP synthase in a primed state for the terminal assembly step-motor module formation.
Abstract Oxidants have a profound impact on biological systems in physiology and under pathological conditions. Oxidative post-translational modifications of protein thiols are well-recognized as a readily occurring alteration of proteins. Changes in protein thiol redox state can modify the function of proteins and thus can control cellular processes. However, chronic oxidative stress causes oxidative damage to proteins with detrimental consequences for cellular function and organismal health. The development of techniques enabling the site-specific and quantitative assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early oxidation targets in key biological processes governing the de novo production of proteins, folding, and protein degradation. Comparison to existing datasets reveals evolutionary conservation of early oxidation targets. To facilitate accessibility and cross-species comparison of the experimental data obtained, we created the OxiAge Database, a free online tool for the research community that integrates current datasets on thiol redoxomes in aged yeast, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster , and mouse Mus musculus . The database can be accessed through an interactive web application at http://oxiage.ibb.waw.pl .
Abstract The autophagy-flux-promoting protein TFG ( Trk-fused gene ) is up-regulated during B cell differentiation into plasma cells and supports survival of CH12 B cells. We hypothesized that quantitative proteomics analysis of CH12 tfg KO B cells with intact or blocked autophagy-lysosome flux (via NH 4 Cl) will identify mechanisms of TFG-dependent autophagy, plasma cell biology and B cell survival. Analysis of CH12WT B cells in the presence of NH 4 Cl will identify proteins whose presence is continuously regulated by lysosomes independent of TFG. We determined hundreds of proteins to be controlled by TFG and/or NH 4 Cl. Notably, NH 4 Cl treatment alone increased the abundance of a cluster of cytosolic and mitochondrial translational proteins while it also reduced a number of proteins. Within the B cell relevant protein pool, BCL10 was reduced, while JCHAIN was increased in CH12 tfg KO B cells. Furthermore, TFG regulated the abundance of transcription factors, such as JUNB, metabolic enzymes, such as the short-chain fatty acid activating enzyme ACOT9 or the glycolytic enzyme ALDOC. Gene ontology enrichment analysis revealed that TFG-regulated proteins localized to mitochondria and membrane-bounded organelles. Due to these findings we performed shotgun lipidomics of glycerophospholipids, uncovering that a particular phosphatidylethanolamine (PE) species, 32:0 PE, which lipidates LC3 most efficiently, was less abundant while phosphatidylglycerol (PG) was more abundant in CH12 tfg KO B cells. In line with the role of PG as precursor for Cardiolipin (CL), the CL content was higher in CH12 tfg KO B cells and addition of PG liposomes to B cells increased the amount of CL. We propose a role for TFG in B cell activation and plasma cell biology via regulation of proteins involved in germinal center and plasma cell development, such as BCL10 or JCHAIN, as well as in lipid homeostasis, mitochondria and metabolism.
This chapter contains sections titled: Introduction Ionization Principles Matrix-Assisted Laser Desorption/Ionization (MALDI) Electrospray Ionization Mass Spectrometric Instrumentation Protein Identification Strategies Quantitative Mass Spectrometry for Comparative and Functional Proteomics Metabolic Labeling Approaches 15N Labeling Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Chemical Labeling Approaches Chemical Isotope Labeling at the Protein Level Stable Isotope Labeling at the Peptide Level Quantitative MS for Deciphering Protein–Protein Interactions Conclusions
Significance Growth of mitochondria and their transmission during cytokinesis depends on protein import from the cytosol as well as on the faithful replication and segregation of their genomes. Here, we show that in the early diverging eukaryote Trypanosoma brucei , a single mitochondrial outer membrane protein controls both the assembly of the master protein translocase as well as the inheritance of the single-unit mitochondrial genome. Although the protein is unique to trypanosomatids, it shows functional similarity to an outer membrane protein of yeast. The concept that a single protein mediates the assembly of the major protein translocase and the transmission of mitochondrial DNA might therefore be conserved in other eukaryotes.
