Rosa Ma Dégano

A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica is more resistant to high copper concentrations thanSaccharomyces cerevisiae. This differential tolerance to copper ions has been observed in several strains arising from non-related isolates. To investigate the molecular basis of this resistance, we obtained several copper-sensitive mutants. By complementation of one of them, we isolated the YlCRF1 gene encoding for a copper-binding transcription factor of 411 amino acids homologous to ScAce1p, CgAmt1p, and ScMac1p. Naturally occurring copper-sensitive strains lack the CRF1 allele. TheYlCRF1 transcript is not induced by the addition of copper to the medium. Gene disruption demonstrated thatYlCRF1 is responsible for a 4- to 5-fold increase in Y. lipolytica copper tolerance. We further show that strain Δcrf1 is more sensitive to cadmium but not to other metals. The role of YlCrf1p as a copper-sensitive transcription factor is supported by the finding that the protein is immunolocalized in the nucleus during growth in copper-supplemented but not in copper-free medium. However, in contrast to the S. cerevisiae strain mutated in the metallothionein transcription activator ACE1, Y. lipolytica strain Δcrf1 is still able to increase metallothionein (MTP) mRNA levels in response to copper addition.CRF1 deletion does not affect superoxide dismutase (SOD) activity either. Our data suggest the existence of one or more different target genes for Crf1p, other than MTP orSOD1, and support its role as a novel copper-responsive transcription factor involved in metal detoxification.

Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project

A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of combining -omics for a comprehensive characterization of specific biological systems.

NAPPA as a real new method for protein microarray generation

Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224‐antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B‐CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down‐regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC‐proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC‐MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.

In-Depth Proteomic Characterization of Classical and Non-Classical Monocyte Subsets

Monocytes are bone marrow-derived leukocytes that are part of the innate immune system. Monocytes are divided into three subsets: classical, intermediate and non-classical, which can be differentiated by their expression of some surface antigens, mainly CD14 and CD16. These cells are key players in the inflammation process underlying the mechanism of many diseases. Thus, the molecular characterization of these cells may provide very useful information for understanding their biology in health and disease. We performed a multicentric proteomic study with pure classical and non-classical populations derived from 12 healthy donors. The robust workflow used provided reproducible results among the five participating laboratories. Over 5000 proteins were identified, and about half of them were quantified using a spectral counting approach. The results represent the protein abundance catalogue of pure classical and enriched non-classical blood peripheral monocytes, and could serve as a reference dataset of the healthy population. The functional analysis of the differences between cell subsets supports the consensus roles assigned to human monocytes.

Evaluation Strategies of Nanomaterials Toxicity

The revolutionary development of nanoscience during the last years has increased the number of studies in the field to evaluate the toxicity and risk levels. The design of different nanomaterials together with biological components has implemented the advances in biomedicine. Specifically, nanoparticles seem to be a promising platform due to their features, including nanoscale dimensions and physical and chemical characteristics than can be modified in function of the final application. Herein, we review the main studies realized with nanoparticles in order to understand and characterize the cellular uptake mechanisms involved in biocompatibility, toxicity, and how they alter the biological processes to avoid disease progression.

A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells

A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next-Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig. Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches. For this purpose, an in–house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.