Josep Villanueva

Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors

Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyzatrifolii larvae in homozygote tomato lines expressing high levels (#62;1 the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect.

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato1: cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5 region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.A partial cDNA clone for the potato wound‐inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)‐treated potato leaves. The full 5′ region of the cDNA was obtained through a RACE‐PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N‐terminal signal peptide, a 27 residue N‐terminal pro‐region, the 39 residue mature PCI protein, and a 7 residue C‐terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C‐terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C‐terminal pro‐domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Unconventional Secretion is a Major Contributor of Cancer Cell Line Secretomes

A challenge in achieving optimal management of cancer is the discovery of secreted biomarkers that represent useful surrogates for the disease and could be measured noninvasively. Because of the problems encountered in the proteomic interrogation of plasma, secretomes have been proposed as an alternative source of tumor markers that might be enriched with secreted proteins relevant to the disease. However, secretome analysis faces analytical challenges that interfere with the search for true secreted tumor biomarkers. Here, we have addressed two of the main challenges of secretome analysis in comparative discovery proteomics. First, we carried out a kinetics experiment whereby secretomes and lysates of tumor cells were analyzed to monitor cellular viability during secretome production. Interestingly, the proteomic signal of a group of secreted proteins correlated well with the apoptosis induced by serum starvation and could be used as an internal cell viability marker. We then addressed a second challenge relating to contamination of serum proteins in secretomes caused by the required use of serum for tumor cell culture. The comparative proteomic analysis between cell lines labeled with SILAC showed a number of false positives coming from serum and that several proteins are both in serum and being secreted from tumor cells. A thorough study of secretome methodology revealed that under optimized experimental conditions there is a substantial fraction of proteins secreted through unconventional secretion in secretomes. Finally, we showed that some of the nuclear proteins detected in secretomes change their cellular localization in breast tumors, explaining their presence in secretomes and suggesting that tumor cells use unconventional secretion during tumorigenesis. The unconventional secretion of proteins into the extracellular space exposes a new layer of genome post-translational regulation and reveals an untapped source of potential tumor biomarkers and drug targets.

Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back‐exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site‐directed mutants of different conformational stability. In this work, we show that by using MALDI‐TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.

Batch effects correction improves the sensitivity of significance tests in spectral counting-based comparative discovery proteomics

Shotgun proteomics has become the standard proteomics technique for the large-scale measurement of protein abundances in biological samples. Despite quantitative proteomics has been usually performed using label-based approaches, label-free quantitation offers advantages related to the avoidance of labeling steps, no limitation in the number of samples to be compared, and the gain in protein detection sensitivity. However, since samples are analyzed separately, experimental design becomes critical. The exploration of spectral counting quantitation based on LC-MS presented here gathers experimental evidence of the influence of batch effects on comparative proteomics. The batch effects shown with spiking experiments clearly interfere with the biological signal. In order to minimize the interferences from batch effects, a statistical correction is proposed and implemented. Our results show that batch effects can be attenuated statistically when proper experimental design is used. Furthermore, the batch effect correction implemented leads to a substantial increase in the sensitivity of statistical tests. Finally, the applicability of our batch effects correction is shown on two different biomarker discovery projects involving cancer secretomes. We think that our findings will allow designing and executing better comparative proteomics projects and will help to avoid reaching false conclusions in the field of proteomics biomarker discovery.

Monitoring the expression and purification of recombinant proteins by MALDI-TOF mass spectrometry

1Samples coming from biologic sources usually contain several contaminants that interfere seriously with Mass Spectrometry (MS) measurements. In this paper we report the application of MALDI-TOF MS to monitor recombinant protein expression and purification. The technique is based on the use of a C18 resin to clean and concentrate proteins in batch. The utility of this method is demonstrated for samples coming from different bacterial cultures expressing secreted and intracellular proteins ranging from 4 to 53 kDa. MALDI-TOF MS of peptide and proteins can be accomplished directly from complex bacterial cultures or from any purification step in a few minutes using the conventional stainless steel sample targets, allowing for a nearly instantaneous monitoring of the nature and integrity of recombinant expression products.

Ligand Screening by Exoproteolysis and Mass Spectrometry in Combination With Computer Modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.

Circulating pEGFR is a candidate response biomarker of cetuximab therapy in colorectal cancer.

Purpose: The lack of secreted biomarkers measurable by noninvasive tests hampers the development of effective targeted therapies against cancer. Our hypothesis is that cetuximab (an anti-EGFR mAb) induces a specific secretome in colorectal cancer cells that could be exploited for biomarker discovery. Experimental Design: Considering the strong correlation between mutated KRAS and a lack of response to cetuximab therapy, we addressed whether performing secretome-based proteomics on isogenic colorectal cancer cells sharing the KRAS mutations found on patients would yield candidate-secreted biomarkers useful in the clinical setting. Because 2D culture did not optimally model the sensitivity/resistance to cetuximab observed in colorectal cancer patients, we moved to 3D spheroids, developing a methodology for both cell-based assays and quantitative proteomics. Results: A large comparative quantitative proteomic analysis of the 3D secretomes of colorectal cancer isogenic cells treated with cetuximab uncovered an EGFR pathway-centric secretome found only when cells grow in 3D. The validation of the secretome findings in plasma of colorectal cancer patients, suggests that phosphorylated-EGFR (pEGFR) is a candidate-secreted biomarker of response to cetuximab. Conclusions: We have proved that 3D spheroids from colorectal cancer cells generate secretomes with a drug-sensitivity profile that correlates well with patients with colorectal cancer, illustrating molecular connections between intracellular and extracellular signaling. Furthermore, we show how the secretion of pEGFR is associated with the sensitivity of colorectal cancer cells to cetuximab and the response of patients with colorectal cancer to the drug. Our work could allow the noninvasive monitoring of anti-EGFR treatment in patients with colorectal cancer. Clin Cancer Res; 20(24); 6346–56. ©2014 AACR.

An effect size filter improves the reproducibility in spectral counting-based comparative proteomics ☆

UNLABELLEDnThe microarray community has shown that the low reproducibility observed in gene expression-based biomarker discovery studies is partially due to relying solely on p-values to get the lists of differentially expressed genes. Their conclusions recommended complementing the p-value cutoff with the use of effect-size criteria. The aim of this work was to evaluate the influence of such an effect-size filter on spectral counting-based comparative proteomic analysis. The results proved that the filter increased the number of true positives and decreased the number of false positives and the false discovery rate of the dataset. These results were confirmed by simulation experiments where the effect size filter was used to evaluate systematically variable fractions of differentially expressed proteins. Our results suggest that relaxing the p-value cut-off followed by a post-test filter based on effect size and signal level thresholds can increase the reproducibility of statistical results obtained in comparative proteomic analysis. Based on our work, we recommend using a filter consisting of a minimum absolute log2 fold change of 0.8 and a minimum signal of 2-4 SpC on the most abundant condition for the general practice of comparative proteomics. The implementation of feature filtering approaches could improve proteomic biomarker discovery initiatives by increasing the reproducibility of the results obtained among independent laboratories and MS platforms.nnnBIOLOGICAL SIGNIFICANCEnQuality control analysis of microarray-based gene expression studies pointed out that the low reproducibility observed in the lists of differentially expressed genes could be partially attributed to the fact that these lists are generated relying solely on p-values. Our study has established that the implementation of an effect size post-test filter improves the statistical results of spectral count-based quantitative proteomics. The results proved that the filter increased the number of true positives whereas decreased the false positives and the false discovery rate of the datasets. The results presented here prove that a post-test filter applying a reasonable effect size and signal level thresholds helps to increase the reproducibility of statistical results in comparative proteomic analysis. Furthermore, the implementation of feature filtering approaches could improve proteomic biomarker discovery initiatives by increasing the reproducibility of results obtained among independent laboratories and MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

Monitoring disappearance of monomers and generation of resistance to proteolysis during the formation of the activation domain of human procarboxypeptidase A2 (ADA2h) amyloid fibrils by matrix-assisted laser-desorption ionization–time-of-flight-MS

The term amyloidosis is used to represent a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. One of the several questions that remain unclear regarding the process of amyloid fibril formation is related to the status of the protein when such a process begins. Protein engineering is one of the selected approaches to study amyloidosis. Characterization of many variants of a protein can give information about why a soluble protein aggregates to form fibrils. In the present study, we report information on the conformational changes that precede the formation of fibrils, monitored by the complementary use of exoproteolysis and matrix-assisted laser-desorption ionization-time-of-flight-MS. This is a novel application of an easy and fast approach. In addition, we used it to evaluate the ability of the model protein ADA2h (activation domain of human procarboxypeptidase A2) and their mutants to generate amyloid fibrils. It could be a useful test to screen protein variants and to study to what extent some physicochemical parameters affect fibrillogenesis.