- Publication type
- Meeting Abstract MeSH
The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α- and β-subunits of succinyl-CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α- and β-SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α- and β-SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α- nor β-SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain-containing membrane-associated protein and tetraspanin family surface protein, as well as soluble-secreted β-amylase-1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α- and β-SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.
It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome.
- MeSH
- Aldehyde Oxidoreductases chemistry metabolism MeSH
- Alcohol Dehydrogenase chemistry metabolism MeSH
- Escherichia coli metabolism MeSH
- Protein Conformation MeSH
- Models, Molecular MeSH
- Porins chemistry metabolism MeSH
- Escherichia coli Proteins chemistry metabolism MeSH
- Ribosomes chemistry genetics metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Breast cancer is the most common malignancy among females worldwide. Recent studies have shown extra-ribosomal roles of the moonlight ribosomal proteins in the development of human cancers. Accurate quantification of the gene expression level is based on the selection of the reference genes whose expression is independent of cancer properties and patient's characteristics. The aim of this study was the evaluation of the expression level of a previously proposed ribosomal protein as moonlight, L13a (RPL13A), in breast cancer samples and their adjacent tissues. Its association with genes of known roles in developing cancers was also investigated. Traditionally used housekeeping genes were selected and their expression was analyzed in 80 surgically excised breast tissue specimens (40 tumors and 40 tumor-adjacent tissues) by applying three software tools including GeNorm, NormFinder, and BestKeeper to select the most stable reference genes. Then, mRNA expression levels of RPL13A and p53 were evaluated. Additionally, protein expression levels of RPL13A were measured. It was demonstrated that PUM1 and ACTB are the most reliable reference genes and RPL13A is the least stable gene. There was a positive correlation between RPL13A and p53 mRNA expression levels in all the tumor samples. Moreover, significant downregulation of RPL13A expression levels was revealed in HER2+ tumor samples compared to HER2- ones. There was also a marked decrease in p53 mRNA expression levels in HER2+ tumor subtypes. Our results suggest that there is a probable relationship between RPL13A decreased expression with p53 and HER2/neu expression in the breast cancer.
- MeSH
- Genes, p53 MeSH
- Humans MeSH
- Breast Neoplasms * MeSH
- Prognosis MeSH
- Ribosomal Proteins MeSH
- Genes, Tumor Suppressor MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Evaluation Study MeSH
Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel β-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K(m) value of 0.88 mmol/L, a k(cat) value of 212/min, and a k(cat)/K(m) value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl(3), CuCl(2), and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.
- MeSH
- Bacteria classification enzymology genetics isolation & purification MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- beta-Glucosidase chemistry genetics metabolism MeSH
- Phylogeny MeSH
- Kinetics MeSH
- Lipase chemistry genetics metabolism MeSH
- Metagenome MeSH
- Molecular Sequence Data MeSH
- Soil Microbiology MeSH
- Amino Acid Sequence MeSH
- Sequence Alignment MeSH
- Enzyme Stability MeSH
- Substrate Specificity MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.
- MeSH
- Bacteria enzymology pathogenicity MeSH
- Bacterial Infections microbiology prevention & control MeSH
- Bacterial Proteins metabolism MeSH
- Bacterial Vaccines immunology MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases immunology metabolism MeSH
- Humans MeSH
- Proteins metabolism MeSH
- Protein Binding MeSH
- Virulence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
SIGNIFICANCE: Hexokinases are key enzymes that are responsible for the first reaction of glycolysis, but they also moonlight other cellular processes, including mitochondrial redox signaling regulation. Modulation of hexokinase activity and spatiotemporal location by reactive oxygen and nitrogen species as well as other gasotransmitters serves as the basis for a unique, underexplored method of tight and flexible regulation of these fundamental enzymes. Recent Advances: Redox modifications of thiols serve as a molecular code that enables the precise and complex regulation of hexokinases. Redox regulation of hexokinases is also used by multiple parasites to cause widespread and severe diseases, including malaria, Chagas disease, and sleeping sickness. Redox-active molecules affect each other, and the moonlighting activity of hexokinases provides another feedback loop that affects the cellular redox status and is hijacked in malignantly transformed cells. CRITICAL ISSUES: Several compounds affect the redox status of hexokinases in vivo. These include the dehydroascorbic acid (oxidized form of vitamin C), pyrrolidinium porrolidine-1-carbodithioate (contraceptive), peroxynitrite (product of ethanol metabolism), alloxan (a glucose analog), and isobenzothiazolinone ebselen. However, very limited information is available regarding which amino acid residues in hexokinases are affected by redox signaling. Except in cases of monogenic diabetes, direct evidence is absent for disease phenotypes that are associated with variations within motifs that are susceptible to redox signaling. FUTURE DIRECTIONS: Further studies should address the propensity of hexokinases and their disease-associated variants to participate in redox regulation. Robust and straightforward proteomic methods are needed to understand the context and consequences of hexokinase-mediated redox regulation in health and disease.
- MeSH
- Hexokinase metabolism MeSH
- Humans MeSH
- Mitochondria metabolism MeSH
- Oxidation-Reduction MeSH
- Signal Transduction MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.
- MeSH
- Glutamate-Ammonia Ligase MeSH
- Licensure MeSH
- Humans MeSH
- Mice MeSH
- Lung Neoplasms * MeSH
- Carcinoma, Non-Small-Cell Lung * MeSH
- Cell Proliferation MeSH
- Cell Cycle Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.
Functional explanations for loud calling in nocturnal primates include territorial or sexual advertisement, maintenance of cohesiveness, and group coordination. It is generally accepted that loud calls of lesser galagos (genus Galago) are used for territorial advertisement and long-distance spacing. Field studies suggest that they are uttered at dusk and dawn, when the animals leave and reunite at their sleeping sites. However, empirical validation of these inferences is lacking. We conducted 16-night-long acoustic monitoring of a northern lesser galago (G. senegalensis) population in Senegal and quantified the occurrence of loud calls throughout the night. We hypothesized that significantly more of these calls would be emitted at dusk and dawn if they were used for territorial advertisement and long-distance spacing. This hypothesis was only partially supported, as we found an asymmetrical distribution of loud calls, which significantly increased only before and at dawn. The finding that the relatively early increase in vocal activity was not directly related to approaching and entering sleeping sites suggests that the northern lesser galagos' loud calls differ in function from reassembly calls described for other species of nocturnal primates. Furthermore, the early onset cannot be explained by changes in the intensity of sunlight, moonlight or starlight, which suggests that a different stimulus, most likely internal, elicits early-morning calling behavior in northern lesser galagos.
- MeSH
- Acoustics MeSH
- Circadian Rhythm MeSH
- Galago physiology MeSH
- Light MeSH
- Vocalization, Animal * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Senegal MeSH
