Stefania Orrù

Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

ABSTRACT The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Analysis of the Ribosomal Protein S19 Interactome

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Axinellins A and B: New Proline‐Containing Antiproliferative Cyclopeptides from the Vanuatu Sponge Axinella carteri

Two new bioactive cyclopeptides, named axinellins A (1) and B (2) have been isolated from the marine sponge Axinella carteri. Their structure elucidation was based on two-dimensional (2D) NMR (500 MHz) as well as HRFABMS and ESMS/MS data. All amino acid residues derived from axinellins A and B were found to possess L configuration at Cα by HPLC analysis of their FDAA derivatives (Marfeys method). The amino acid sequence of 1 and 2 was established on the basis of tandem mass spectrometry data (ESMS/MS) and on 1H-1H through-space connectivities observed in NOESY and ROESY spectra. Axinellins A (1) and B (2) exhibited moderate in vitro antitumor activity against human broncopulmonary non-small-cell-lung-carcinoma lines (NSCLC-N6) with IC50 values of 3.0 and 7.3 μg/ml, respectively.

Biological and Nutritional Properties of Palm Oil and Palmitic Acid: Effects on Health

A growing body of evidence highlights the close association between nutrition and human health. Fat is an essential macronutrient, and vegetable oils, such as palm oil, are widely used in the food industry and highly represented in the human diet. Palmitic acid, a saturated fatty acid, is the principal constituent of refined palm oil. In the last few decades, controversial studies have reported potential unhealthy effects of palm oil due to the high palmitic acid content. In this review we provide a concise and comprehensive update on the functional role of palm oil and palmitic acid in the development of obesity, type 2 diabetes mellitus, cardiovascular diseases and cancer. The atherogenic potential of palmitic acid and its stereospecific position in triacylglycerols are also discussed.

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo‐2 and HCT‐116. Putative CSCs were separated from non‐CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133– cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β‐catenin pathway, and we demonstrated a direct cause–effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium‐dependent enzymes that catalyze a post‐translational modification of proteins through the formation of ε‐(γ‐glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well‐characterized biotinylated transglutaminase substrates as affinity probes: 5–(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino‐ and acyl‐donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.

Purification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds.

The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an M(r) of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51-44% identical positions) with other plant LTP2s previously described.

SRp20: an overview of its role in human diseases.

Alternative splicing in mRNA maturation has emerged as a major field of study also because of its implications in various diseases. The SR proteins play an important role in the regulation of this process. Evidence indicates that SRp20 (SFSR3), the smallest member of the SR protein family, is involved in numerous biological processes. Here we review the state-of-the-art of knowledge about the SR proteins, in particular SRp20, in terms of its function and misregulation in human diseases including cancer also in view of its potential as a therapeutic target.

Transcription factor TBX1 overexpression induces downregulation of proteins involved in retinoic acid metabolism: a comparative proteomic analysis.

TBX1 haploinsufficiency is considered a major contributor to the del22q11.2/DiGeorge syndrome (DGS) phenotype. We have used proteomic tools to look at all the major proteins involved in the TBX1-mediated pathways in an attempt to better understand the molecular interactions instrumental to its cellular functions. We found more than 90 proteins that could be targeted by TBX1 through different mechanisms. The most interesting observation is that overexpression of TBX1 results in down-regulation of two proteins involved in retinoic acid metabolism.

Androgen receptor signaling induced by supraphysiological doses of dihydrotestosterone in human peripheral blood lymphocytes

Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand‐activated transcription factors. AR binds specific DNA elements, known as androgen–response elements. Testosterone, the main male sexual hormone, binds AR directly and indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. Anabolic androgenic steroids are frequently detected in the urine of doped athletes; their consumption is also growing among sport amateurs and adolescents. The effects of androgens can differ depending on the target cells and/or tissues. To gain insight into transcription activation mechanisms of AR, we investigated AR protein signaling in human peripheral blood lymphocytes treated with supraphysiological doses of DHT. We performed a comparative proteomic analysis and we identified about 30 differentially expressed proteins. At least five species contained a consensus androgen–response elements sequence in the promoter region of related coding genes. The analysis also revealed that high doses of DHT activate the drug detoxification process, could stimulate an increase in cell motility and exert a prosurvival effect rather than an apoptotic one.