Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. METHODS: We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt's and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment' synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. RESULTS: We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. CONCLUSIONS: The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Malassezia pachydermatis from brown bear: A comprehensive analysis reveals novel genotypes and distribution of all detected variants in domestic and wild animals.

Malassezia pachydermatis (phylum Basidiomycota, class Malasseziomycetes) is a zoophilic opportunistic pathogen with recognized potential for invasive infections in humans. Although this pathogenic yeast is widespread in nature, it has been primarily studied in domestic animals, so available data on its genotypes in the wild are limited. In this study, 80 yeast isolates recovered from 42 brown bears (Ursus arctos) were identified as M. pachydermatis by a culture-based approach. MALDI-TOF mass spectrometry (MS) was used to endorse conventional identification. The majority of samples exhibited a high score fluctuation, with 42.5% of isolates generating the best scores in the range confident only for genus identification. However, the use of young biomass significantly improved the identification of M. pachydermatis at the species confidence level (98.8%). Importantly, the same MALDI-TOF MS efficiency would be achieved regardless of colony age if the cut-off value was lowered to ≥1.7. Genotyping of LSU, ITS1, CHS2, and ß-tubulin markers identified four distinct genotypes in M. pachydermatis isolates. The most prevalent among them was the genotype previously found in dogs, indicating its transmission potential and adaptation to distantly related hosts. The other three genotypes are described for the first time in this study. However, only one of the genotypes consisted of all four loci with bear-specific sequences, indicating the formation of a strain specifically adapted to brown bears. Finally, we evaluated the specificity of the spectral profiles of the detected genotypes. MALDI-TOF MS exhibited great potential to detect subtle differences between all M. pachydermatis isolates and revealed distinct spectral profiles of bear-specific genotypes.

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines.

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

Intra-Species Variations of Bioactive Compounds of Two Dictyota Species from the Adriatic Sea: Antioxidant, Antimicrobial, Dermatological, Dietary, and Neuroprotective Potential.

The marine environment has a significant impact on life on Earth. Organisms residing in it are vital for the ecosystem but also serve as an inexhaustible source of biologically active compounds. Herein, the biodiversity of two brown seaweeds, Dictyota dichotoma and Dictyota fasciola from the Adriatic Sea, was evaluated. The aim of the study was the determination of differences in compound composition while comparing their activities, including antioxidant, antimicrobial, and enzyme inhibition, in connection to human digestion, dermatology, and neurological disorders. Chemical analysis revealed several terpenoids and steroids as dominant molecules, while fucoxanthin was the main identified pigment in both algae. D. dichotoma had higher protein, carbohydrate, and pigment content. Omega-6 and omega-3 fatty acids were identified, with the highest amount of dihomo-γ-linolenic acid and α-linolenic acid in D. dichotoma. Antimicrobial testing revealed a dose-dependent inhibitory activity of methanolic fraction against Escherichia coli and Staphylococcus aureus. Moderate antioxidant activity was observed for both algae fractions, while the dietary potential was high, especially for the D. fasciola dichloromethane fraction, with inhibition percentages of around 92% for α-amylase and 57% for pancreatic lipase at 0.25 mg/mL. These results suggest that Dictyota species might be a potent source of naturally derived agents for obesity and diabetes.

Phosphoproteome Dynamics of Streptomyces rimosus during Submerged Growth and Antibiotic Production.

Streptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium. IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Ancient Origins of Cytoskeletal Crosstalk: Spectraplakin-like Proteins Precede the Emergence of Cortical Microtubule Stabilization Complexes as Crosslinkers.

Adhesion between cells and the extracellular matrix (ECM) is one of the prerequisites for multicellularity, motility, and tissue specialization. Focal adhesions (FAs) are defined as protein complexes that mediate signals from the ECM to major components of the cytoskeleton (microtubules, actin, and intermediate filaments), and their mutual communication determines a variety of cellular processes. In this study, human cytoskeletal crosstalk proteins were identified by comparing datasets with experimentally determined cytoskeletal proteins. The spectraplakin dystonin was the only protein found in all datasets. Other proteins (FAK, RAC1, septin 9, MISP, and ezrin) were detected at the intersections of FAs, microtubules, and actin cytoskeleton. Homology searches for human crosstalk proteins as queries were performed against a predefined dataset of proteomes. This analysis highlighted the importance of FA communication with the actin and microtubule cytoskeleton, as these crosstalk proteins exhibit the highest degree of evolutionary conservation. Finally, phylogenetic analyses elucidated the early evolutionary history of spectraplakins and cortical microtubule stabilization complexes (CMSCs) as model representatives of the human cytoskeletal crosstalk. While spectraplakins probably arose at the onset of opisthokont evolution, the crosstalk between FAs and microtubules is associated with the emergence of metazoans. The multiprotein complexes contributing to cytoskeletal crosstalk in animals gradually gained in complexity from the onset of metazoan evolution.

Biotechnological Enhancement of Probiotics through Co-Cultivation with Algae: Future or a Trend?

The diversity of algal species is a rich source of many different bioactive metabolites. The compounds extracted from algal biomass have various beneficial effects on health. Recently, co-culture systems between microalgae and bacteria have emerged as an interesting solution that can reduce the high contamination risk associated with axenic cultures and, consequently, increase biomass yield and synthesis of active compounds. Probiotic microorganisms also have numerous positive effects on various aspects of health and represent potent co-culture partners. Most studies consider algae as prebiotics that serve as enhancers of probiotics performance. However, the extreme diversity of algal organisms and their ability to produce a plethora of metabolites are leading to new experimental designs in which these organisms are cultivated together to derive maximum benefit from their synergistic interactions. The future success of these studies depends on the precise experimental design of these complex systems. In the last decade, the development of high-throughput approaches has enabled a deeper understanding of global changes in response to interspecies interactions. Several studies have shown that the addition of algae, along with probiotics, can influence the microbiota, and improve gut health and overall yield in fish, shrimp, and mussels aquaculture. In the future, such findings can be further explored and implemented for use as dietary supplements for humans.

The Landscape of Signaling Pathways and Proteasome Inhibitors Combinations in Multiple Myeloma.

Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an extreme genetic heterogeneity that poses great challenges for its successful treatment. Due to antibody overproduction, MM cells depend on the precise regulation of the protein degradation systems. Despite the success of PIs in MM treatment, resistance and adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. To this end, the use of rational combinatorial treatments might allow lowering the dose of inhibitors and therefore, minimize their side-effects. Even though the suppression of different cellular pathways in combination with proteasome inhibitors have shown remarkable anti-myeloma activities in preclinical models, many of these promising combinations often failed in clinical trials. Substantial progress has been made by the simultaneous targeting of proteasome and different aspects of MM-associated immune dysfunctions. Moreover, targeting deranged metabolic hubs could represent a new avenue to identify effective therapeutic combinations with PIs. Finally, epigenetic drugs targeting either DNA methylation, histone modifiers/readers, or chromatin remodelers are showing pleiotropic anti-myeloma effects alone and in combination with PIs. We envisage that the positive outcome of patients will probably depend on the availability of more effective drug combinations and treatment of early MM stages. Therefore, the identification of sensitive targets and aberrant signaling pathways is instrumental for the development of new personalized therapies for MM patients.

Novel arsenic hyper-resistant bacteria from an extreme environment, Crven Dol mine, Allchar, North Macedonia.

Novel hyper-resistant bacteria were isolated from the Crven Dol mine (Allchar, North Macedonia), arsenic-rich extreme environment. Bacteria were recovered from a secondary mineral mixture, an alteration of hydrothermal realgar rich in arsenates (pharmacolite, hornesite, and talmessite). The sample was recovered from the dark part of the mine at 28 m depth. Three bacterial strains and a bacterial consortium were isolated for their capacity to survive exposure to 32 g/L (209 mM) of arsenite, and 176 g/L (564 mM) of arsenate. The 16S rRNA gene analysis identified bacterial isolates as Stenotrophomonas sp. and two Microbacterium spp. This analysis also revealed that bacterial consortium comprise two Bacteriodetes exhibiting similarity to Olivibacter ginsengisoli and to uncultured bacterium, and one γ-proteobacteria with similarity to Luteimonas sp. Among all isolates Stenotrophomonas sp. exhibited the highest tolerance to As compound as well as the capacity to accumulate As inside the cells. Analysis of genes involved in As-resistance showed that recovered isolates possess the genes encoding the ArsB, Acr3(1) and Acr3(2) proteins, indicating that at least a part of their resistance could be ascribed to As-efflux systems described in isolates obtained from human-polluted environments.

Leitmotif: protein motif scanning 2.0.

MOTIVATION: Motif-HMM (mHMM) scanning has been shown to possess unique advantages over standardly used sequence-profile search methods (e.g. HMMER, PSI-BLAST) since it is particularly well-suited to discriminate proteins with variations inside conserved motifs (e.g. family subtypes) or motifs lacking essential residues (false positives, e.g. pseudoenzymes). RESULTS: In order to make mHMM widely accessible to a broader scientific community, we developed Leitmotif, an mHMM web application with many parametrization options easily accessible through intuitive interface. Substantial improvement of performance (ROC scores) was obtained by using two novel parameters. To the best of our knowledge, Leitmotif is the only available mHMM application. AVAILABILITY AND IMPLEMENTATION: Leitmotif is freely available at https://leitmotif.irb.hr. CONTACT: sinisa@heuristika.hr or ivan.vujaklija@fer.hr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An effective approach for annotation of protein families with low sequence similarity and conserved motifs: identifying GDSL hydrolases across the plant kingdom.

BACKGROUND: The massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom. RESULTS: Motif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through the graphical user interface ( http://compbio.math.hr/ ). CONCLUSIONS: Our results show that scanning with a carefully parameterized motif-HMM is an effective approach for annotation of protein families with low sequence similarity and conserved motifs. The results of this study expand current knowledge and provide new insights into the evolution of the large GDSL-lipase family in land plants.

Exploring Actinobacteria assemblages in coastal marine sediments under contrasted Human influences in the West Istria Sea, Croatia.

The exploration of marine Actinobacteria has as major challenge to answer basic questions of microbial ecology that, in turn, will provide useful information to exploit Actinobacteria metabolisms in biotechnological processes. The ecological functions performed by Actinobacteria in marine sediments are still unclear and belongs to the most burning basic questions. The comparison of Actinobacteria communities inhabiting marine sediments that are under the influence of different contamination types will provide valuable information in the adaptation capacities of Actinobacteria to colonize specific ecological niche. In the present study, the characterization of different Actinobacteria assemblages according to contamination type revealed the ecological importance of Actinobacteria for maintaining both general biogeochemical functions through a "core" Actinobacteria community and specific roles associated with the presence of contaminants. Indeed, the results allowed to distinguish Actinobacteria genera and species operational taxonomic units (OTUs) able to cope with the presence of either (i) As, (ii) metals Ni, Fe, V, Cr, and Mn, or (iii) polycyclic aromatic hydrocarbons (PAHs) and toxic metals (Hg, Cd, Cu, Pb, and Zn). Such observations highlighted the metabolic capacities of Actinobacteria and their potential that should be taken into consideration and advantage during the implementation of bioremediation processes in marine ecosystems.

Structure-function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation.

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Šresolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.