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    • ABC super family proteins are found in all forms of

    2024-03-15

    ABC super family proteins are found in all forms of life and ABC-F subfamily of proteins are involved in cellular processes such as translational control and antibiotic resistance by ribosomal protection [3], [5]. The Mtb ABC proteins that do not contain transmembrane domains have not been studied [6], [7]. Three mycobacterial fatty acyltransferases and a lipase that are likely involved in the triacylglycerol biosynthetic pathway [19] are located in the immediate vicinity of Rv2477c and its orthologs in mycobacteria (Fig. 1A). The potential involvement of Rv2477c in membrane and storage lipid biosynthesis in Mtb needs to be investigated in future studies. Rv2477c displays homology to the EttA protein in E. coli that is involved in the energy-dependent control of translational elongation. E. coli cells lacking EttA exhibited a severe fitness defect during long-term stationary phase [8]. The overall amino SB408124 receptor sequence identity of Rv2477c and the E. coli EttA was nearly 57% and the “LSGGE” ABC signature motifs and catalytic glutamate residues at positions 188 and 470 on EttA were conserved in Rv2477c. Further studies are needed on the potential interaction of Rv2477c with the Mtb ribosome. A microarray study of Mtb genes showed that the transcripts of Rv2477c were upregulated in response to exposure to ofloxacin [9]. Recently, a single-nucleotide polymorphism in the gene encoding Rv2477c was linked to antibiotic resistance in Mtb[10]. The Rv2477c protein shows significant homologies with bacterial ABC-F subfamily proteins like Vga(A) involved in antibiotic resistance in Staphylococcus aureus (Fig. 1B). Since Vga(A) is inhibited by antibiotics that target protein translation [16], [17], it is likely that Rv2477c also belongs to the antibiotic resistance group of proteins. We observed that the ATPase activity of Rv2477c is also inhibited by tetracycline and the macrolide erythromycin which are antibiotics that target protein translation (Table 2). The ATPase activity was stimulated to the highest level by Mn2+ followed by Co2+ and Mg2+ (Fig. 2C). Manganese/cobalt-dependent ATPases have been characterized in mycobacteria and in archaea [20], [21]. Mn2+ levels are thought to be highest in slow growing or stationary-phase bacteria [22]. Since Mtb enters a non-replicating or stationary phase when it encounters growth-limiting stresses inside the human body [23], [24], [25], the roles of Mn2+-regulated enzymes such as Rv2477c might be relevant to pathogenesis. The isoelectric point of Rv2477c is 4.95 [11]. Remarkably, the maximum ATPase activity was observed at pH 5.2 and very little activity was observed at pH 4.0 and pH 7.0 or higher (Fig. 2D). Mtb encounters acidic conditions inside the macrophage in the human body [26]. Therefore, the observed optimal pH for the activity of Rv2477c might be relevant to physiological conditions experienced by Mtb during pathogenesis. The glutamate residues in the Walker B motifs of ABC proteins have been shown to be involved in the second ATP hydrolysis step and their substitutions to glutamine result in the mutant proteins having a very low level of ATPase activity [14], [15]. We observed similar results with the Rv2477c-EQ2 mutant protein confirming that the purified Rv2477c protein exhibits a typical ATPase catalytic mechanism (Fig. 3C). Further studies on the role of Rv2477c in Mtb when the pathogen encounters stress conditions that mimic those inside the human body are needed for a deeper understanding of this ABC-F protein family member.
    Acknowledgements This work was supported by funding to J.D. from the Purdue Research Foundation, the Department of Biology and the Office of Sponsored Programs of the Indiana University-Purdue University, Fort Wayne, IN. The authors declare no conflict of interest.
    Introduction The vacuolar H+-ATPase (V-ATPase) is an electrogenic proton pump that acidifies the lumen of intra- and extracellular compartments. More than 30years have passed after the first discovery of the enzyme from yeast vacuole membranes [1]. Its presence in yeast indicates that the enzyme is required for basic cellular physiology; indeed, the enzyme is considered as having a housekeeping function. V-ATPase is now known to be present in both uni- and multicellular organisms, including plants and animals. The enzyme is distributed in various cellular locations; specifically, in endomembrane organelles such as the lysosomes, endosomes, the Golgi apparatus, secretory granules, and coated vesicles. They are also present in the plasma membrane of specialized cell types including osteoclasts as well as epithelial cells in the kidney, male genital tract, and ocular ciliary bodies. The acidic pH generated by V-ATPase is required for diverse cellular processes including endosomal ligand–receptor dissociation, hormone concentration, lysosomal degradation, and acid secretion (reviewed in [2]). These findings indicate that V-ATPase is supporting higher order physiological processes in the multicellular organisms, in addition to the fundamental cellular function.