Biomedical subjects

Enrique Calvo

Publications and source records attributed to Enrique Calvo.

12 recordsLinked to original sources

PubMed2007

Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.

Alkanes↗

PubMed2006

Human Cdc14A reverses CDK1 phosphorylation of Cdc25A on serines 115 and 320.

Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.

Amino Acid Sequence↗

PubMed2006

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research.

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Botrytis↗

PubMed2006

Mass spectrometry technologies for proteomics.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.

Mass Spectrometry↗

PubMed2006

Proteomic and transcriptional characterization of aromatic degradation pathways in Rhodoccocus sp. strain TFB.

Rhodococcus sp. strain TFB is a versatile gram-positive bacterium able to grow on a wide variety of aromatic compounds as carbon and energy sources. Since the strain is refractory to genetic analysis, a proteomic approach was used to study the metabolic pathways involved in the catabolism of such compounds by analyzing differentially induced proteins. The most marked difference was observed when the proteome profiles of phthalate-grown cells were compared with those cultured in the presence of tetralin- or naphthalene, suggesting that different metabolic pathways are involved in the degradation of mono- and polyaromatic compounds. Comparison with the proteome of glucose-grown cells indicated that each pathway was specifically induced by the corresponding aromatic compound. A combination of proteomics and molecular biology led to the identification of 14 proteins (65-80% identical to known Pht proteins) that describe a complete pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid. Chaperonins were also induced in phthalate-grown cells, indicating that growth on this compound induces a stress response. Absence of catabolite repression by glucose was observed by both transcriptional and proteome analysis, suggesting that Rhodococcus sp. strain TFB may have advantages over other tightly regulated strains in bioremediation.

Amino Acid Sequence↗

PubMed2006

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex.

The human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.

Amino Acid Substitution↗

PubMed2005

Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer.

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Acetylation↗

PubMed2005

Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry.

Genome analyses have revealed that the Gram-positive bacterial species Listeria monocytogenes and L. innocua contain a large number of genes encoding surface proteins predicted to be covalently bound to the cell wall (41 and 34, respectively). The function of most of these proteins is unknown and they have not even been identified biochemically. Here, we report the first characterization of the Listeria cell wall proteome using a nonelectrophoretic approach. The material analyzed consisted of a peptide mixture obtained from a cell wall extract insoluble in boiling 4% SDS. This extract, containing peptidoglycan (intrinsically resistant to proteases) and strongly associated proteins, was digested with trypsin in a solution with 0.01% SDS, used to favor protein digestion throughout the peptidoglycan. The resulting complex peptide mixture was fractionated and analyzed by two-dimensional nanoliquid chromatography coupled to ion-trap mass spectrometry. A total of 30 protein species were unequivocally identified in cell wall extracts of the genome strains L. monocytogenes EGD-e (19 proteins) and L. innocua CLIP11262 (11 proteins). Among them, 20 proteins bearing an LPXTG motif recognized for covalent anchoring to the peptidoglycan were identified. Other proteins detected included peptidoglycan-lytic enzymes, a penicillin-binding protein, and proteins bearing an NXZTN motif recently proposed to direct protein anchoring to the peptidoglycan. The marked sensitivity of the method makes it highly attractive in the post-genome era for defining the cell wall proteome in any bacterial species. This information will be useful to study novel protein-peptidoglycan associations and to rapidly identify new targets in the surface of important bacterial pathogens.

Cell Wall↗

PubMed2005

Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis.

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria by cleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif is LPXTG, which is cleaved between the T and G residues. In this study, a non-gel proteomic approach was used to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA and SrtB. Material containing peptidoglycan and strongly associated proteins was purified from sortase-defective mutants, digested with trypsin, and the resulting peptide mixture analysed by two-dimensional nano-liquid chromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycan metabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surface proteins, Lmo2185 and Lmo2186, were identified only when SrtB was active. The analysis of the peptides identified in these proteins suggests that SrtB of L. monocytogenes may recognize two different sorting motifs, NXZTN and NPKXZ. Taken together, these data demonstrate that non-gel proteomics is a powerful technique to rapidly identify sortase substrates and to gain insights on potential sorting motifs.

Amino Acid Sequence↗

PubMed2003

[Intrahospital mortality after discharge from the ICU (hidden mortality) in patients who required mechanical ventilation].

BACKGROUND AND OBJECTIVE: Our goal was to determine the hidden mortality (HM) in patients who underwent an episode of mechanical ventilation (MV). We also analyzed the factors associated with an increase in the risk of hidden mortality. PATIENTS AND METHOD: Prospective cohort study. Patients admitted to an ICU who required MV and who were monitored until their discharge from hospital. We performed a multivariate study with a logistic regression model including all the variables that were present in a univariate analysis p < 0.20. RESULTS: Forty-one of the 215 patients who were discharged from the ICU died when they were admitted to hospital, which represents a hidden mortality rate of 19% (CI 95% 11%-27%). A mean period of 9 days elapsed between discharge from the ICU and patient's death, with 25% of patients dying within the first two days. Commonest cause of death was respiratory failure (37%). Factors independently associated with an increase in the risk of hidden mortality were (values expressed as adjusted odds ratio (CI 95%): age > 74 years 1.15 (1.01 to 1.26) (p = 0.02); APACHE II > 29 1.14 (1.01 to 1.27) (p = 0.04); reason for MV being coma 1.21 (1.07 to 1.37) (p = 0.002); reason for MV being cardiopulmonary arrest 1.28 (1.18 to 1.68) (p < 0.001); tracheotomy in ICU 1.31 (1.19 to 1.68) (p < 0.001) and stay in the ICU longer than 16 days 1.35 (1.01 to1.70) (p = 0.04). CONCLUSIONS: An important number of patients discharged from the ICU after an episode of MV die in hospital. Risk factors associated with an increased risk of death in hospital identify a group of patients who, after excluding those with non-cardiopulmonary resuscitation orders, would possibly benefit from high surveillance or intermediate care units.

APACHE↗

PubMed2002

Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron.

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs.

Animals↗