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.

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.

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.

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.

Chemoproteomic fishing identifies arzanol as a positive modulator of brain glycogen phosphorylase.

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.

Plasticizers used in food-contact materials affect adipogenesis in 3T3-L1 cells.

Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25 µM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein ß (Cebpß) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis.

Biomolecular proteomics discloses ATP synthase as the main target of the natural glycoside deglucoruscin.

Extracts of Ruscus aculeatus are a rich source of bioactive steroidal glycosides, such as ruscogenins which are reported to act against chronic venous disorders. Nowadays, several preparations of its roots, commonly used in traditional medicine, are on the market as food supplements for health care and maintenance. Although spirostanol deglucoruscin is one of the main metabolites in these extracts, literature reports about its pharmacological profile are scarce. In this paper, a multi-disciplinary approach, based on chemical proteomics, molecular modelling and bio-organic assays, has been used to disclose the whole interactome of deglucoruscin and the F0-F1 ATP synthase complex has been found as its main target.

In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus).

Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERß1, ERß2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERß, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish.

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells.

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells.

Environmental pollutants directly affect the liver X receptor alpha activity: Kinetic and thermodynamic characterization of binding.

Liver X receptor is a ligand-activated transcription factor, which is mainly involved in cholesterol homeostasis, bile acid and triglycerides metabolism, and, as recently discovered, in the glucose metabolism by direct regulation of liver glucokinase. Its modulation by exogenous factors, such as drugs, industrial by-products, and chemicals is documented. Owing to the abundance of these synthetic molecules in the environment, and to the established target role of this receptor, a number of representative compounds of phthalate, organophosphate and fibrate classes were tested as ligands/modulators of human liver X receptor, using an integrated approach, combining an in silico molecular docking technique with an optical SPR biosensor binding study. The compounds of interest were predicted and proved to target the oxysterols-binding site of human LXRα with measurable binding kinetic constants and with affinities ranging between 4.3 × 10(-7) and 4.3 × 10(-8)M. Additionally, non-cytotoxic concentration of these chemicals induced relevant changes in the LXRα gene expression levels and other target genes (SREBP-1c and LGK) in human liver hepatocellular carcinoma cell line (HepG2), as demonstrated by q-RT-PCR.

Pseudomonas fluorescens Pf-5 genome-wide mutant screen for resistance to the antimicrobial peptide alfalfa snakin-1.

Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.

Arene-ruthenium(II) acylpyrazolonato complexes: apoptosis-promoting effects on human cancer cells.

A series of ruthenium(II) arene complexes with the 4-(biphenyl-4-carbonyl)-3-methyl-1-phenyl-5-pyrazolonate ligand, and related 1,3,5-triaza-7-phosphaadamantane (PTA) derivatives, has been synthesized. The compounds have been characterized by NMR and IR spectroscopy, ESI mass spectrometry, elemental analysis, and X-ray crystallography. Antiproliferative activity in four human cancer cell lines was determined by MTT assay, yielding dose- and cancer cell line-dependent IC50 values of 9-34 µM for three hexamethylbenzene-ruthenium complexes, whereas the other metal complexes were much less active. Apoptosis was the mechanism involved in the anticancer activity of such compounds. In fact, the hexamethylbenzene-ruthenium complexes activated caspase activity, with consequent DNA fragmentation, accumulation of pro-apoptotic proteins (p27, p53, p89 PARP fragments), and the concomitant down-regulation of antiapoptotic protein Bcl-2. Biosensor-based binding studies indicated that the ancillary ligands were critical in determining the DNA binding affinities, and competition binding experiments further characterized the nature of the interaction.

Binding of CD157 protein to fibronectin regulates cell adhesion and spreading.

CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.

Mechanistic insights on petrosaspongiolide M inhibitory effects on immunoproteasome and autophagy.

The proteasome, a complex multimeric structure strictly implicated in cell protein degradation, has gained the status of privileged drug target since its functional involvement in relevant pathways ruling the cell life, such as cell cycle, transcription and protein quality control, and the recent marketing of bortezomib as proteasome inhibitor for anti-cancer therapy. The marine γ-hydroxybutenolide terpenoid petrosaspongiolide M has been recently discovered as new proteasome inhibitor through a chemical proteomic approach and in cell biological assays. In this study a deep investigation has been carried out on the molecular mechanism of interaction of petrosaspongiolide M with the immunoproteasome, a proteasomal variant mainly involved in the immune responses. The results define a picture in which petrosaspongiolide M exerts its inhibitory activity by binding the active sites in the inner core of the immunoproteasome and/or covalently linking a Lys residue at the proteasome core/11S activator particle interface. Moreover, petrosaspongiolide M is also able to impair autophagy, a complementary pathway involved in protein degradation and cross-talking with the proteasome system. On this basis, petrosaspongiolide M could represent an interesting molecule for its propensity to modulate intracellular proteolysis through a dual inhibition of the immunoproteasome and autophagy.

Wild type and mutant amyloid precursor proteins influence downstream effects of proteasome and autophagy inhibition.

Cells rely on complementary proteolytic pathways including the ubiquitin-proteasome system and autophagy to maintain proper protein degradation. There is known to be considerable interplay between them, whereby the loss of one clearance system results in compensatory changes in other proteolytic pathways of the cell. Disturbances in proteolysis are known to occur in Alzheimer's disease, and potentially contribute to neurophysiological and neurodegenerative processes. Currently, few data are available on how the presence of wild type and mutant amyloid precursor protein (APPwt and APPmut) potentially alters the reciprocal interplay between the different intracellular proteolytic pathways. This study used human SH-SY5Y neuronal cell lines, and SH-SY5Y transfected with either APPwt or APPmut (valine-to-glycine substitution at position 717), in order to explore if the presence of APPwt or APPmut altered the downstream effects of pharmacological proteasome or autophagy inhibition. The occurrence of APPwt or APPmut was observed to disturb proteasome or autophagy activities upon treatment with proteasome inhibitors or authophagy inhibitors. Interestingly, APPwt and APPmut expression was observed to significantly and robustly enhance the induction in cathepsin B following the administration of an established proteasome inhibitor. The presence of APPwt and APPmut also significantly reduced the elevation in ubiquitinated proteins following proteasome inhibitor treatments. Our data strongly suggest that APP is able to affect the downstream effects of protease inhibition in neural cells including enhancement of cathepsin B activity, with these changes in cathepsin B significantly and inversely related to the levels of ubiquitinated protein.

Chemical proteomics-driven discovery of oleocanthal as an Hsp90 inhibitor.

Hsp90, a key target in cancer therapy, has been identified as the main partner of oleocanthal, an olive oil bioactive compound. A combination of chemical and biological assays disclosed its mechanism of action at the molecular level.

Ghrelin induces apoptosis in colon adenocarcinoma cells via proteasome inhibition and autophagy induction.

Ghrelin is a metabolism-regulating hormone recently investigated for its role in cancer survival and progression. Controversially, ghrelin may act as either anti-apoptotic or pro-apoptotic factor in different cancer cells, suggesting that the effects are cell type dependent. Limited data are currently available on the effects exerted by ghrelin on intracellular proteolytic pathways in cancer. Both the lysosomal and the proteasomal systems are fundamental in cellular proliferation and apoptosis regulation. With the aim of exploring if the proteasome and autophagy may be possible targets of ghrelin in cancer, we exposed human colorectal adenocarcinoma cells to ghrelin. Preliminary in vitro fluorimetric assays evidenced for the first time a direct inhibition of 20S proteasomes by ghrelin, particularly evident for the trypsin-like activity. Moreover, 1 µM ghrelin induced apoptosis in colorectal adenocarcinoma cells by inhibiting the ubiquitin-proteasome system and by activating autophagy, with p53 having an "interactive" role.