Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics.

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

A label free chemoproteomic-based platform to disclose cannabidiol molecular mechanism of action on chronic myelogenous leukemia cancer cells.

The discovery of the interactome of cannabidiol (CBD), a non-psychoactive cannabinoid from Cannabis sativa L., has been here performed on chronic myelogenous leukemia cancer cells, using an optimized chemo-proteomic stage, which links Drug Affinity Responsive Target Stability with Limited Proteolysis Multiple Reaction Monitoring approaches. The obtained results showed the ability of CBD to target simultaneously some potential protein partners, corroborating its well-known poly-pharmacology activity. In human chronic myelogenous leukemia K562 cancer cells, the most fascinating protein partner was identified as the 116 kDa U5 small nuclear ribonucleoprotein element called EFTUD2, which fits with the spliceosome complex. The binding mode of this oncogenic protein with CBD was clarified using mass spectrometry-based and in silico analysis.

Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform.

Tatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3-methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and ß-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism.

Skin Gene Expression Profiles in Systemic Sclerosis: From Clinical Stratification to Precision Medicine.

Systemic sclerosis, also known as scleroderma or SSc, is a condition characterized by significant heterogeneity in clinical presentation, disease progression, and response to treatment. Consequently, the design of clinical trials to successfully identify effective therapeutic interventions poses a major challenge. Recent advancements in skin molecular profiling technologies and stratification techniques have enabled the identification of patient subgroups that may be relevant for personalized treatment approaches. This narrative review aims at providing an overview of the current status of skin gene expression analysis using computational biology approaches and highlights the benefits of stratifying patients upon their skin gene signatures. Such stratification has the potential to lead toward a precision medicine approach in the management of SSc.

Screening and Analysis of Possible Drugs Binding to PDGFRα: A Molecular Modeling Study.

The platelet-derived growth factor receptor (PDGFR) is a membrane tyrosine kinase receptor involved in several metabolic pathways, not only physiological but also pathological, as in tumor progression, immune-mediated diseases, and viral diseases. Considering this macromolecule as a druggable target for modulation/inhibition of these conditions, the aim of this work was to find new ligands or new information to design novel effective drugs. We performed an initial interaction screening with the human intracellular PDGFRα of about 7200 drugs and natural compounds contained in 5 independent databases/libraries implemented in the MTiOpenScreen web server. After the selection of 27 compounds, a structural analysis of the obtained complexes was performed. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were also performed to understand the physicochemical properties of identified compounds to increase affinity and selectivity for PDGFRα. Among these 27 compounds, the drugs Bafetinib, Radotinib, Flumatinib, and Imatinib showed higher affinity for this tyrosine kinase receptor, lying in the nanomolar order, while the natural products included in this group, such as curcumin, luteolin, and epigallocatechin gallate (EGCG), showed sub-micromolar affinities. Although experimental studies are mandatory to fully understand the mechanisms behind PDGFRα inhibitors, the structural information obtained through this study could provide useful insight into the future development of more effective and targeted treatments for PDGFRα-related diseases, such as cancer and fibrosis.

Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five ß-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

A Proteomic Platform Unveils the Brain Glycogen Phosphorylase as a Potential Therapeutic Target for Glioblastoma Multiforme.

In the last few years, several efforts have been made to identify original strategies against glioblastoma multiforme (GBM): this requires a more detailed investigation of the molecular mechanism of GBM so that novel targets can be identified for new possible therapeutic agents. Here, using a combined biochemical and proteomic approach, we evaluated the ability of a blood-brain barrier-permeable 2,3-benzodiazepin-4-one, called 1g, to interfere with the activity and the expression of brain glycogen phosphorylase (PYGB) on U87MG cell line in parallel with the capability of this compound to inhibit the cell growth and cycle. Thus, our results highlighted PYGB as a potential therapeutic target in GBM prompting 1g as a capable anticancer drug thanks to its ability to negatively modulate the uptake and metabolism of glucose, the so-called "Warburg effect", whose increase is considered a common feature of cancer cells in respect of their normal counterparts.

PDGF/PDGFR: A Possible Molecular Target in Scleroderma Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous disorder of the connective tissue characterized by vascular alterations, immune/inflammatory manifestations, and organ fibrosis. SSc pathogenesis is complex and still poorly understood. Therefore, effective therapies are lacking and remain nonspecific and limited to disease symptoms. In the last few years, many molecular and cellular mediators of SSc fibrosis have been described, providing new potential options for targeted therapies. In this review: (i) we focused on the PDGF/PDGFR pathway as key signaling molecules in the development of tissue fibrosis; (ii) we highlighted the possible role of stimulatory anti-PDGFRα autoantibodies in the pathogenesis of SSc; (iii) we reported the most promising PDGF/PDGFR targeting therapies.

The macronuclear genome of the Antarctic psychrophilic marine ciliate Euplotes focardii reveals new insights on molecular cold adaptation.

The macronuclear (MAC) genomes of ciliates belonging to the genus Euplotes species are comprised of numerous small DNA molecules, nanochromosomes, each typically encoding a single gene. These genomes are responsible for all gene expression during vegetative cell growth. Here, we report the analysis of the MAC genome from the Antarctic psychrophile Euplotes focardii. Nanochromosomes containing bacterial sequences were not found, suggesting that phenomena of horizontal gene transfer did not occur recently, even though this ciliate species has a substantial associated bacterial consortium. As in other euplotid species, E. focardii MAC genes are characterized by a high frequency of translational frameshifting. Furthermore, in order to characterize differences that may be consequent to cold adaptation and defense to oxidative stress, the main constraints of the Antarctic marine microorganisms, we compared E. focardii MAC genome with those available from mesophilic Euplotes species. We focussed mainly on the comparison of tubulin, antioxidant enzymes and heat shock protein (HSP) 70 families, molecules which possess peculiar characteristic correlated with cold adaptation in E. focardii. We found that α-tubulin genes and those encoding SODs and CATs antioxidant enzymes are more numerous than in the mesophilic Euplotes species. Furthermore, the phylogenetic trees showed that these molecules are divergent in the Antarctic species. In contrast, there are fewer hsp70 genes in E. focardii compared to mesophilic Euplotes and these genes do not respond to thermal stress but only to oxidative stress. Our results suggest that molecular adaptation to cold and oxidative stress in the Antarctic environment may not only be due to particular amino acid substitutions but also due to duplication and divergence of paralogous genes.

Sequence and Properties of Cagein, a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus.

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as "basal-body cages." The major protein comprising Euplotes cages has been previously identified and termed "cagein." Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear "nanochromosome" of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.

An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation.

Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.

Dissection of the gut microbiota in mothers and children with chronic Trichuris trichiura infection in Pemba Island, Tanzania.

BACKGROUND: Soil-transmitted helminthiases are important neglected tropical diseases that result in a notably high number of disability-adjusted life years worldwide. Characterizing the interactions between the human intestinal microbiome and helminths is of interest in the development of alternative treatments that do not rely on chemotherapeutics and do not lead to drug resistance. METHODS: We recruited and obtained fecal samples from 32 pairs of mothers and children on Pemba Island and monitored their intestinal microbiota using 16S rRNA gene sequencing. RESULTS: We observed that microbial changes occur in the gut microbiota of infected mothers and children. Some short-chain fatty acid (SCFA)-producing bacteria and carbohydrate-degrading bacteria exhibited lower abundance in the infected individuals. Potentially pathogenic Campylobacter and proinflammatory Methanobrevibacter in infected mothers and opportunistic Enterococcus in infected children exhibited greater abundance. CONCLUSIONS: Our findings could reveal the microbiota profiling in T. trichiura-infected individuals, indicate the potential roles of key microbiota in the host and aid to the development of novel strategies to control T. trichiura infection.

Transcriptomic responses to silver nanoparticles in the freshwater unicellular eukaryote Tetrahymena thermophila.

Currently, silver nanoparticles (AgNPs) are being increasingly used as biocides in various consumer products and if released in the environment they can affect non-target organisms. Therefore, understanding the toxicity mechanism is crucial for both the design of more efficient nano-antimicrobials and for the design of nanomaterials that are biologically and environmentally benign throughout their life-cycle. Here, the ciliate Tetrahymena thermophila was used to elucidate the mechanisms of action of AgNPs by analysing the gene expression profile by RNA-seq and the transcriptomic effects of AgNPs were compared to those induced by soluble silver salt, AgNO3. Exposure to AgNPs at sublethal concentrations for 24 h induced phagocytosis, transport pathways, response to oxidative stress, glutathione peroxidase activity, response to stimulus, oxidation-reduction, proteolysis, and nitrogen metabolism process. Based on gene set enrichment analysis (GSEA), some biological processes appeared targets of both toxicants. In addition to many similarities in affected genes, some effects were triggered only by NPs, like phagocytosis, glutathione peroxidase activity, response to stimulus, protein phosphorylation and nitrogen metabolism process. This research provides evidence that AgNPs compared to AgNO3 at the same concentration of dissolved silver ions dysregulate a higher number of cellular pathways. These findings confirm that AgNPs can induce toxicity not only due to soluble silver ions released from the particles but also to particle intrinsic features.

Horizontal gene transfer and silver nanoparticles production in a new Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii.

We isolated a novel bacterial strain from a prokaryotic consortium associated to the psychrophilic marine ciliate Euplotes focardii, endemic of the Antarctic coastal seawater. The 16S rDNA sequencing and the phylogenetic analysis revealed the close evolutionary relationship to the Antarctic marine bacterium Marinomonas sp. BSw10506 and the sub antarctic Marinomonas polaris. We named this new strain Marinomonas sp. ef1. The optimal growth temperature in LB medium was 22 °C. Whole genome sequencing and analysis showed a reduced gene loss limited to regions encoding for transposases. Additionally, five genomic islands, e.g. DNA fragments that facilitate horizontal gene transfer phenomena, were identified. Two open reading frames predicted from the genomic islands coded for enzymes belonging to the Nitro-FMN-reductase superfamily. One of these, the putative NAD(P)H nitroreductase YfkO, has been reported to be involved in the bioreduction of silver (Ag) ions and the production of silver nanoparticles (AgNPs). After the Marinomonas sp. ef1 biomass incubation with 1 mM of AgNO3 at 22 °C, we obtained AgNPs within 24 h. The AgNPs were relatively small in size (50 nm) and had a strong antimicrobial activity against twelve common nosocomial pathogenic microorganisms including Staphylococcus aureus and two Candida strains. To our knowledge, this is the first report of AgNPs biosynthesis by a Marinomonas strain. This biosynthesis may play a dual role in detoxification from silver nitrate and protection from pathogens for the bacterium and potentially for the associated ciliate. Biosynthetic AgNPs also represent a promising alternative to conventional antibiotics against common pathogens.

Carbamoyl-Phosphate Synthase 1 as a Novel Target of Phomoxanthone A, a Bioactive Fungal Metabolite.

Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

Crellastatin A, a PARP-1 Inhibitor Discovered by Complementary Proteomic Approaches.

Crellastatin A, a cytotoxic sulfated bis-steroid isolated from the Vanuatu Island marine sponge Crella sp., was selected as an interesting probe for a comprehensive proteomic analysis directed at the characterization of its protein interactors. Given its peculiar structural features, A was submitted to a mass spectrometry-based drug affinity responsive target stability (DARTS) assay combined with (targeted-limited proteolysis-multiple reaction monitoring (t-LiP MRM), rather than a classical affinity purification strategy. Poly-ADP-ribose-polymerase-1 (PARP-1) emerged as the main crellastatin A cellular partner. This result was confirmed by both biochemical and in silico analyses. Further in vitro biological assays highlighted an interesting crellastatin A inhibitory activity on PARP-1.

Draft Genome Sequence of a New Pseudomonas sp. Strain, ef1, Associated with the Psychrophilic Antarctic Ciliate Euplotes focardii.

We announce here the draft genome sequence of a new Pseudomonas strain, named Pseudomonas sp. strain ef1, associated with the cold-adapted Antarctic ciliate Euplotes focardii The genome sequence is 6,228,167 bp long with a G+C content of 59.7%.

Chemical Proteomics-Guided Identification of a Novel Biological Target of the Bioactive Neolignan Magnolol.

Understanding the recognition process between bioactive natural products and their specific cellular receptors is of key importance in the drug discovery process. In this outline, some potential targets of Magnolol, a natural bioactive compound, have been identified by proteomic approaches. Among them, Importin-ß1 has been considered as the most relevant one. A direct binding between Magnolol and this nuclear chaperone has been confirmed by DARTS and molecular docking, while its influence on Importin-ß1 translocation has been evaluated by in vitro assays.

PDGFA in Cashmere Goat: A Motivation for the Hair Follicle Stem Cells to Activate.

The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation.