José Ignacio Casal

Proteome analysis of membrane fractions in colorectal carcinomas by using 2D-DIGE saturation labeling.

Because of its high content in receptors and signaling proteins, the analysis of membrane fractions is critical for the study of neoplastic diseases as colorectal cancer. Here, we have used the new saturation labeling for 2D-DIGE analysis of the membrane proteome of colorectal cancer mucosal tissues. Samples from 6 patients (tumoral and normal paired biopsies) were included in this study. Twelve analytical gels were performed and considered for the quantitative study and statistical analysis. A spot pattern analysis, by using an unsupervised clustering algorithm, allowed the classification of the samples according to similar expression patterns in tumoral and normal samples. Those proteins whose expression changed significantly (Students t-test, p < 0.05) were further digested and characterized by mass spectrometry. Among the differentially expressed proteins: annexin A2, annexin A4, annexin A5, annexin A7, lamin B, calponin 1 and VDAC were analyzed by immunohistochemistry using tissue microarrays. Annexin A2, annexin A4 and VDAC appear as potential markers of interest for colorectal cancer diagnosis and, presumably, therapy. In summary, saturation labeling provides a new and sensitive tool for the analysis of scarce amounts of samples, allowing sample classification and direct identification of deregulated proteins.

Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.

SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue

SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial–mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT–PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). Expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P=0.047 and P=0.014, respectively) and NAT lacking SNAI1 expression (P=0.054 and P=0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r=0.39; P=0.027) and inversely to CDH1 in NAT (r=−0.46; P=0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VDR.

Synthetic Peptide Vaccines: Success at Last

Peptide vaccines would form the ideal ultimate vaccine because they are safe both in production as well as application, easy to handle, store and transport. Peptide vaccines have the advantage that they can be produced in a completely reproducible manner, they are cheap compared to subunit or whole protein vaccines and potentially they can be precisely targeted to meet specific demands. For instance, peptide vaccines may be used to break maternal immunity or raise very specific antibodies for research or diagnostics. Furthermore synthetic peptide vaccines offer the potential possibility for multivalent vaccines and to incorporate adjuvanticity. Notwithsanding numerous attempts only one peptide vaccine has found its way to the market. This peptide vaccine is however not targeted to a pathogen but to a self-protein, Gonadotropin Releasing Hormone (GnRH), and used to immunocastrate production, animals (Hoskinson et al, 1990). In this contribution we shall discuss why peptide vaccines for pathogens, notably viruses, have failed so far, by focusing on Foot-and-Mouth Disease Virus (FMDV). For FMDV substantial attempts have been made to develop a synthetic peptide vaccine. These attempts have failed. However an attempt to develop a peptide vaccine against CPV was successful and may help to learn how failures with other attempts can be overcome.

Colorectal cancer proteomics, molecular characterization and biomarker discovery

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2‐D DIGE, MS and protein microarray technologies, in their different formats, to identify up‐ or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low‐abundant proteins, which may play a key role in CRC diagnostics and progression.

Mapping the antigenic structure of porcine parvovirus at the level of peptides

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Surfing transcriptomic landscapes. A step beyond the annotation of chromosome 16 proteome

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

A combinatorial approach for the design of complementarity-determining region-derived peptidomimetics with in vitro anti-tumoral activity

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH2) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (Kd values in micromolar range) and the parental monoclonal antibodies (Kd values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.

AKT2 interacts with Snail1 in the E-cadherin Promoter

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.

Designing antibodies for the inhibition of gastrin activity in tumoral cell lines

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin‐2 (CCK‐2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK‐2 receptor. The gastrin neutralizing activity assay showed that N‐terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti‐C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody‐based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity.