Laura Villarreal

The Saccharomyces cerevisiae Crs5 Metallothionein metal-binding abilities and its role in the response to zinc overload

Crs5 is a Saccharomyces cerevisiae Metallothionein (MT), non‐homologous to the paradigmatic Cu‐thionein Cup1. Although considered a secondary copper‐resistance agent, we show here that it determines survival under zinc overload in a CUP1‐null background. Its overexpression prevents the deleterious effects exhibited by CUP1‐CRS5‐null cells when exposed to combined Zn/Cu, as it does the mouse MT1 Zn‐thionein, but not Cup1. The detailed characterization of Crs5 in vivo and in vitro Zn(II)‐, Cd(II)‐ and Cu(I)‐binding abilities fully supports its resemblance to mammalian MTs. Hence, Crs5 exhibits a good divalent metal‐binding ability, yielding homometallic, highly chiral and stable Zn and Cd complexes when expressed in media enriched with these metal ions. In Cu‐supplemented cultures, heterometallic Zn,Cu complexes are recovered, unless aeration is kept to a minimum. These features define a Crs5 dual metal‐binding behaviour that is significantly closer to Zn‐thioneins than to Cu‐thioneins. Protein sequence similarities fully support these findings. Overall, a Crs5 function in global metal cell homeostasis, based on its Zn‐binding features, is glimpsed. The comparative evaluation of Crs5 in the framework of MT functional differentiation and evolution allows its consideration as a representative of the primeval eukaryotic forms that progressively evolved to give rise to the Zn‐thionein lineage.

MTO: the second member of a Drosophila dual copper-thionein system.

Drosophila MTO metal binding features were analyzed for comparison with MTN, the paralogous Drosophila metallothionein, and to classify MTO as either zinc‐ or copper‐thionein. This was achieved by a combination of in vivo, in vitro and in silico methodologies. All the results unambiguously classified MTO as a second Drosophila copper‐thionein, putting Drosophila forward as the only metazoan in which any zinc‐thionein has still to be reported. Interestingly, experimental data only showed minor differences in the coordinative behavior of both MTs, but provided a characteristic spectroscopic fingerprint, revealing the possible binding of chloride anions in certain metal‐MTO aggregates.

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.

Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein.

Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one‐copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four‐member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc‐ and copper‐thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C‐terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal‐binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low‐copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships.

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.

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 ☆

UNLABELLED The 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. BIOLOGICAL SIGNIFICANCE Quality 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.

The Zn- and Cd-Clusters of Recombinant Mammalian MT1 and MT4 Metallothionein Domains Include Sulfide Ligands:

Recombinant (E. coll) synthesis of mammalian MT1 and MT4 domains as separate peptides in Zn(II) and Cd(II) enriched growth media has rendered metal complexes containing sulfide anions as additional ligands. The Cd preparations show higher sulfide content than the Zn preparations. Also, the βMT1 and βMT4 fragments exhibit higher sulfide/peptide ratios than the respective α fragments. Titration of Zn3-βMT1 with Cd(II) followed by addition of several sodium sulfide equivalents shows that the Cd(II)-βMT1 species can incorporate sulfide ligands in vitro, with a concomitant evolution of their UV-vis and CD fingerprints to those characteristic of the Cd-S2- chromophores. Current results have also provided full understanding of previous data collected by this group in the characterization of the Cd-βMT1 preparations obtained from large-scale fermentor synthesis by allowing identification of at least 2S2- ligands per Cd-βMT1 species. Furthermore, the results here presented have revealed that synthesis of βMT4 in Cd-supplemented cultures yielded Cd,S2--containing clusters instead of the proposed heterometallic Zn, Cd-βMT4 complexes. Finally, a global evaluation of our results suggests that the higher the Cu-thionein character of a MT peptide, the higher is its tendency to harbor nonproteic ligands (i.e., sulfide anions) when building divalent metal clusters, especially Cd-MT complexes.

Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.