Search

A study on the product inhibition of the NS3 protease of hepatitis C virus (HCV) and on the role of Lys136 in stabilizing the enzyme-product complex is presented. The NS3 protein of HCV contains a serine protease domain which if inactivated shows potential as a way of dealing with the disease. Results show that N-terminal cleavage products bind to the NS3 protease during product inhibition with low micromolar affinities.

The complex formation between NS3 and its peptide cofactor was studied by monitoring enzymatic activity changes and the enzymes' spectroscopic properties as a physicochemical environment function. Results show that complex formation did not change the protein's secondary structure and depends on the glycerol presence in the buffer. This suggests that NS4A is an important enzyme in the protein complex stability.

A homology model of the chymotrypsin-like serine proteinase domain in the NS3 protein of hepatitis C virus was constructed. The model indicated the existence of a tetradentate metal binding site formed by three cysteines and one histidine. Findings revealed that the hepatitis C virus enzyme has a Zn2+ ion with S3N ligation and that the metal is needed for structural integrity and activity of the enzyme. The residues forming the metal binding site were also conserved in the chymotrypsin-like 2A cysteine proteinases of picornaviruses.

The first vibrational study of nitric oxide (NO) bound to an oxidized heme-copper oxidase is reported. Cytochrome cbb3 oxidase from Pseudomonas stutzeri reduces both O2 and NO to H2O and N2O respectively.

Resonance Raman spectroscopy (RR) a technique that enables iron ligand stretching modes to be identified to characterize the NO-bound complex of cytochrome cbb(sub 3) oxidase is described. The existence of a five-coordinate heme b(sub 3) Fe(super 2+)-NO complex was found.

Research has been conducted on the heme-copper cbb (sub)3 cytochrome oxidase. The structural characteristics of the cbb (sub)3 binuclear center have been investigated and the results are reported.

A study on the genetic mechanisms of the hepatitis C virus investigated the interaction of the NS3 protease domain of the virus with its NS4A co-factor peptide to identify factors that influence the binding reaction. It will be shown that complex affinities may differ by nearly three orders of magnitude depending on the physico-chemical conditions under which they are determined. Evidence is also presented for complex stabilization by substrate peptides to the active site of the enzyme.

Presents a homology model that predicts the components of the zinc binding site of the serine protease domain of NS3 protein of hepatitis C virus. Need of zinc for enzymatic activity; Properties of the metal-substituted NS3 proteinases.

Examines the influence of the heme Fe-CO complex of cytochrome cbb[sub 3]-CO at ambient temperature formation on the decay of the transient Cu[sub B]-CO complex. Use of time-resolved fourier transform infrared spectroscopy; Catalyzation of molecular oxygen reduction by the complex; Importance of photodynamics of cbb[sub 3]-CO for elucidating the mechanisms for legal entry/exit in heme-cooper oxidase.

We synthesized new pyrrole and indole derivatives as human carbonic anhydrase (hCA) inhibitors with the potential to inhibit the Wnt/β-catenin signaling pathway. The presence of both N 1-(4-sulfonamidophenyl) and 3-(3,4,5-trimethoxyphenyl) substituents was essential for strong hCA inhibitors. The most potent hCA XII inhibitor 15 ( K i = 6.8 nM) suppressed the Wnt/β-catenin signaling pathway and its target genes MYC, Fgf20, and Sall4 and exhibited the typical markers of apoptosis, cleaved poly(ADP-ribose)polymerase, and cleaved caspase-3. Compound 15 showed strong inhibition of viability in a panel of cancer cells, including colorectal cancer and triple-negative breast cancer cells, was effective against the NCI/ADR-RES DOX-resistant cell line, and restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells. Compound 15 is a novel dual-targeting compound with activity against hCA and Wnt/β-catenin. It thus has a broad targeting spectrum and is an anticancer agent with specific potential in P-glycoprotein overexpressing cell lines.

Despite intensive efforts, no inhibitors of the Wnt/β-catenin signaling pathway have been approved so far for the clinical treatment of cancer. We synthesized novel N -(heterocyclylphenyl)benzenesulfonamides as β-catenin inhibitors. Compounds 5 - 10 showed strong inhibition of the luciferase activity. Compounds 5 and 6 inhibited the MDA-MB-231, HCC1806, and HCC1937 TNBC cells. Compound 9 induced in vitro cell death in SW480 and HCT116 cells and in vivo tumorigenicity of a human colorectal cancer line HCT116. In a co-immunoprecipitation study in HCT116 cells transfected with Myc-tagged T-cell factor 4 (Tcf-4), compound 9 abrogated the association between β-catenin and Tcf-4. The crystallographic analysis of the β-catenin Armadillo repeats domain revealed that compound 9 and Tcf-4 share a common binding site within the hotspot binding region close to Lys508. To our knowledge, compound 9 is the first small molecule ligand of this region to be reported. These results highlight the potential of this novel class of β-catenin inhibitors as anticancer agents., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.

p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

Neuroblastoma is one of the most aggressive solid tumors in the childhood. Therapy resistance to anticancer drugs represents the major limitation to the effectiveness of clinical treatment. To better understand the mechanisms underlying cisplatin resistance, we performed a comparative proteomic study of the human neuroblastoma cell line SH-SY5Y and its cisplatin resistant counterpart by both the classical 2-DE electrophoresis coupled to mass spectrometry and the more innovative label-free nLC-MS(E). The differentially expressed proteins were classified by bioinformatic tools according to their biological functions and their involvement in several cellular processes. Moreover, a meta-mining investigation of protein ontologies was also performed on available data from previously published proteomics studies to highlight the modulation of significant cellular pathways, which may regulate the sensitivity of neuroblastoma to cisplatin. In particular, we hypothesized a major role of the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. Confocal microscopy experiments, enzyme assay, and Western blotting of proteins regulated by Nrf2 provided evidences that this pathway, playing a protective role in normal cells, may represent a potential novel target to control cisplatin resistance in neuroblastoma.

Vav1, whose physiological expression is restricted to hematopoietic system, is one of the signaling proteins up-regulated by all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL)-derived precursors, in which it promotes the overcoming of the differentiation blockade. High levels of tyrosine phosphorylated Vav1 accumulate in differentiating APL-derived cells, suggesting that one or more Vav1 tyrosine residues are involved in neutrophil differentiation of tumoral promyelocytes. Here, we have found that phosphorylation of Vav1 Y174, that is known to regulate Vav1 activity in mature neutrophils, is up-regulated by ATRA in NB4 cells. Nevertheless, this tyrosine residue does not seem crucial for the agonist-induced phenotypical differentiation of APL-derived cells. Mass spectrometry analysis performed on Vav1 from differentiating NB4 cells allowed to identify the highly conserved Y745 residue as a phosphorylated tyrosine that plays crucial roles in the completion of the maturation program of this cell line. In fact, the overexpression of a mutated form of Vav1, in which Y745 was replaced with a phenylalanine, significantly reduced the ATRA-induced CD11b expression and essentially abrogated the differentiation-related acquisition of the migratory capability. Even though the intracellular signaling involving Vav1 phosphorylated in Y745 is unknown, the identification of a tyrosine residue essential for differentiation of tumoral precursors may constitute the basis to identify new specific targets for differentiation therapy of APL.

In the last years, proteomic investigation provided a powerful tool in molecular characterization of complex allergen sources with relevant implications in both diagnosis and immunotherapic treatment of allergies. We followed a proteomic approach to characterize ryegrass (Lolium perenne) pollen, a common cause of seasonal allergic diseases affecting an increasing part of world population. Peptide shotgun experiments performed on nanoLiquid Ultra Pressure Chromatography coupled with fast Q-TOF MS-MS/MS acquisition protocols (MS(E)) and 2-DE immunoblot combined with MALDI-TOF-TOF analysis allowed the detection of all previously identified ryegrass allergens. Comparative analysis of immunoblot highlighted a class of patients characterized by a more complex 2-DE pattern associated with increased levels of IgE antibodies and by higher susceptibility to multiple sensitization toward different allergen sources. Cluster analysis revealed that all these patients recognized profilin, considered the main cross-reactive allergen in grass pollen. Furthermore, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization, such as cyclophilin, fructosyltransferase and legumin-like protein.

Epithelial ovarian cancer is the leading cause of gynecological cancer mortality. Despite good response to surgery and initial chemotherapy, chemoresistance occurrence represents a major obstacle to a successful therapy. To better understand biological mechanisms at the basis of paclitaxel resistance, a comparative proteomic approach based on DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS) was applied to the human epithelial ovarian cancer cell lines A2780 and its paclitaxel resistant counterpart A2780TC1. Most of the differentially expressed proteins between the two cell lines belong to the class of stress response (29%), metabolism (21%), and cell cycle and apoptosis (17%). We focused on proteins which were most strongly modulated by paclitaxel resistance and in particular on the disulphide isomerase ERp57, which may represent a chemoresistance biomarker. ERp57 was found to interact with class III beta-tubulin (TUBB3), involved in paclitaxel resistance in ovarian and other cancers. Moreover, we demonstrated a novel localization of this protein in cytoskeleton and described that ERp57/TUBB3 interaction occurs also in the nuclear compartment and in association with a multimeric complex formed by nucleolin, nucleophosmin, hnRNPK, and mortalin. Our data suggest that ERp57 plays an important role in chemoresistance mechanisms in ovarian cancer by modulating the attachment of microtubules to chromosomes following paclitaxel treatment through its interaction with TUBB3.

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.

Understanding the chemical nature of the nitric oxide (NO) moiety of nitrosylheme copper oxidases is crucial for elucidation of the NO activation process. In the present work, direct resonance Raman spectroscopic observation of both the Fe(2+)-NO and the N-O stretching modes unambiguously establishes the vibrational characteristics of the NO-bound heme moiety in cytochrome cbb(3) from Pseudomonas stutzeri. Addition of NO to fully reduced enzyme causes the rupture of the proximal His-heme b(3) bond resulting in the formation of a five-coordinate heme b(3)(2+)-NO species with nu(Fe-NO) and nu(NO) at 524 and 1679 cm(-1), respectively. The frequencies of the nitrosyl species we detect are very similar to those obtained in other model- and protein heme-NO complexes. To account for this observation, we propose a model describing the oxidation and ligand-binding states in fully reduced cytochrome cbb(3) upon addition of NO.