Streptomyces coelicolor Vesicles: Many Molecules to Be Delivered

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content

Poplar woody root proteome during the transition dormancy-active growth

Woody plants living in temperate climates finely regulate their growth and development in relation to seasonal changes; their transition from vegetative to dormancy phase represents an adaptation to their environment. Events occurring in the shoot during onset/release from dormancy have been largely investigated, whereas in woody roots they remain completely unknown. In recent years, we have been interested in understanding the molecular and physiological events occurring in poplar woody root during release from dormancy. Here, we propose the results of a comparative analysis of the proteome of poplar woody root sampled at different time points: T0 (dormancy condition), T1 (release from dormancy), and T2 (full vegetative condition). This study identified proteins that may be involved in the long-term survival of a dormant root or landmarking a specific time point

NADP+-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

A NADP+ dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (E.C. 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM33 µM,kcat.405 min-1), as it is practically inactive towards trans-4-hydroxy-2-nonenal (HNE) and other HNE-addicted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionyl-nonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power

Cladosporol A stimulates G1-phase arrest of the cell cycle by up-regulation of p21waf1/cip1 expression in human colon carcinoma HT-29 cells

Cladosporols, purified and characterized as secondary metabolites from Cladosporium tenuissimum, display an antifungal activity. In this study, we tested the antiproliferative properties of cladosporol A, the main isoform of this metabolite family, against human cancer cell lines. By assessing cell viability, we found that cladosporol A inhibits the growth of various human colon cancers derived cell lines (HT-29, SW480, and CaCo-2) in a time- and concentrationdependent manner, specifically of HT-29 cells. The reduced cell proliferation was due to a G1-phase arrest, as assessed by fluorescence activated cell sorting analysis on synchronized HT-29 cells, and was associated with an early and robust over-expression of p21waf1/cip1, the well-known cyclin-dependent kinases inhibitor. This suggests that the drug may play a role in the control of cancer cell proliferation. Consistently, cyclin D1, cyclin E, CDK2, and CDK4 proteins were reduced and histone H1-associated CDK2 kinase activity inhibited. In addition to p21waf1/cip1, exposure to 20 mM cladosporol A caused a simultaneous increase of pERK and pJNK, suggesting that this drug activates a circuit that integrates cell cycle regulation and the signaling pathways both involved in the inhibition of cell proliferation. Finally, we showed that the increase of p21waf1/cip1 expression was generated by a Sp1-dependent p53-independent stimulation of its gene transcription as mutagenesis of the Sp1 binding sites located in the p21 proximal promoter abolished induction. To our knowledge, this is the first report showing that cladosporol A inhibits colon cancer cell proliferation by modulating p21waf1/cip1 expression

The small protein SCO2038 controls Streptomyces coelicolor differentiation by modulating tryptophan biosynthesis

Background In Streptomyces coelicolor amino acid metabolism is an important clue of the morphological and physiological differentiation program and, differently from other bacteria, the expression of amino acid biosynthetic genes is not subjected to endproduct negative regulation. In some amino acid biosynthetic gene clusters, such as tryptophan, histidine and proline, small orfs (about 100-300 nucleotides) were identified. These small orfs, such as sco2038, encode proteins whose cellular role have to be elucidated to highlight possible novel and crucial molecular mechanisms controlling amino acid synthesis and, thus, differentiation program. Objectives The aims of this work are: 1. the understanding of the effects exerted by tryptophan on primary metabolism, morphological differentiation and antibiotic production; 2. the study and characterization of the SCO2038 function as modulator of tryptophan biosynthesis. Methods - Differential proteomic analysis based on 2D-DIGE and MS procedures. - SEM analysis. - Generation and characterization of sco2038 mutants - Identification of potential SCO2038 interaction partners by pull down assay coupled with MS identification and Bacterial Adenylate Cyclase Two Hybrid System. - qRT-PCR analysis. Conclusions The obtained results revealed that tryptophan controls the expression of metabolic and regulatory proteins and promotes aerial mycelium formation, spores production and actinorhodin antibiotic biosynthesis. Moreover, the small orf sco2038, encodes a 7 KDa protein playing a key role in modulating tryptophan biosynthesis and thus, morphological differentiation. In the light of these results we propose to rename sco2038 as trpM, the gene encoding the tryptophan biosynthesis Modulator TrpM

SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line

Background: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. Results: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. Conclusion: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

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