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Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"—proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins—vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10−8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall—agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)—were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively. [ABSTRACT FROM AUTHOR]

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-β-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, β-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the β-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for β-glucan biosynthesis involved a β-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the β-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. [ABSTRACT FROM AUTHOR]

eEF1A1 and eEF1A2 are paralogous proteins whose presence in most normal eukaryotic cells is mutually exclusive and developmentally regulated. Often described in the scientific literature under the collective name eEF1A, which stands for eukaryotic elongation factor 1A, their best known activity (in a monomeric, GTP-bound conformation) is to bind aminoacyl-tRNAs and deliver them to the A-site of the 80S ribosome. However, both eEF1A1 and eEF1A2 are endowed with multitasking abilities (sometimes performed by homo- and heterodimers) and can be located in different subcellular compartments, from the plasma membrane to the nucleus. Given the high sequence identity of these two sister proteins and the large number of post-translational modifications they can undergo, we are often confronted with the dilemma of discerning which is the particular proteoform that is actually responsible for the ascribed biochemical or cellular effects. We argue in this review that acquiring this knowledge is essential to help clarify, in molecular and structural terms, the mechanistic involvement of these two ancestral and abundant G proteins in a variety of fundamental cellular processes other than translation elongation. Of particular importance for this special issue is the fact that several de novo heterozygous missense mutations in the human EEF1A2 gene are associated with a subset of rare but severe neurological syndromes and cardiomyopathies. [ABSTRACT FROM AUTHOR]

In recent years, cyclic guanosine 3′,5′-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules—BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP. [ABSTRACT FROM AUTHOR]

Plants as sessile organisms face daily environmental challenges and have developed highly nuanced signaling systems to enable suitable growth, development, defense, or stalling responses. Moonlighting proteins have multiple tasks and contribute to cellular signaling cascades where they produce additional variables adding to the complexity or fuzziness of biological systems. Here we examine roles of moonlighting kinases that also generate 3′,5′-cyclic guanosine monophosphate (cGMP) in plants. These proteins include receptor like kinases and lipid kinases. Their guanylate cyclase activity potentiates the development of localized cGMP-enriched nanodomains or niches surrounding the kinase and its interactome. These nanodomains contribute to allosteric regulation of kinase and other molecules in the immediate complex directly or indirectly modulating signal cascades. Effects include downregulation of kinase activity, modulation of other members of the protein complexes such as cyclic nucleotide gated channels and potential triggering of cGMP-dependent degradation cascades terminating signaling. The additional layers of information provided by the moonlighting kinases are discussed in terms of how they may be used to provide a layer of fuzziness to effectively modulate cellular signaling cascades. [ABSTRACT FROM AUTHOR]