Biomedical subjects

Patricia Alfonso

Publications and source records attributed to Patricia Alfonso.

7 recordsLinked to original sources

PubMed2006

Protein pE120R of African swine fever virus is post-translationally acetylated as revealed by post-source decay MALDI mass spectrometry.

Post-translational modification of proteins is a key regulatory event in many cellular processes. African swine fever virus (ASFV) is a large DNA virus that contains about 150 open reading frames (ORF) which encode for more than 150 polypeptides, most of them without assigned function. Two-dimensional gel electrophoresis (2DE) followed by Post-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (PSD-MALDI-MS) revealed that ASFV protein pE120R, essential for virus transport from assembly sites to plasma membranes, is acetylated at the N-terminal Ala residue during infection. To our knowledge, this is the first acetylated ASFV protein described and this modification might be relevant to ASFV life cycle since many viruses use the acetylation signaling pathway as a primary target for viral proteins after infection.

Acetylation↗

PubMed2006

Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts.

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.

Animals↗

PubMed2005

An efficient expression system for the production of functionally active human LKB1.

Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.

AMP-Activated Protein Kinase Kinases↗

PubMed2005

Oxidation of carboxyamidomethyl cysteine may add complexity to protein identification.

Protein identification by interrogation of databases requires a comprehensive compilation of modified amino acids forms. Here, we describe the chemical oxidation of carboxyamidomethyl cysteine to the sulfoxide and sulfone forms, species that may add more complexity to peptide analyses. They can be easily distinguished by tandem mass spectrometry (MS/MS) due to their characteristic pattern of side chain neutral eliminations either from the parent ion or ion series that generate dehydroalanine as detected by MS(3). This finding was supported by the MS(n) spectra recorded for a peptide isolated from a mixture of tryptic peptides and for a derivatized/oxidized synthetic peptide with a different sequence. These modifications and their diagnostic neutral losses should be included in the list of chemical modifications and in algorithms designed for the automatic sequencing of peptides and database searching.

Acetamides↗

PubMed2005

Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis.

The identification of specific protein markers for colorectal cancer would provide the basis for early diagnosis and detection, as well as clues for understanding the molecular mechanisms governing cancer progression. In this report, we describe the proteomic analysis of the samples of colorectal cancer corresponding to seven patients. We have used the highly sensitive two-dimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) for the identification of proteins differentially expressed in tumoral and neighboring normal mucosa. We have detected differences in abundance of 52 proteins with statistical variance of the tumor versus normal spot volume ratio within the 95th confidence level (Student's t-test; p < 0.05). Forty-one out of 52 analyzed proteins were unambiguously identified by matrix-assisted laser desorption/ionization-time of flight MS coupled with database interrogation as being differentially expressed in colorectal cancer. An ontology analysis of these proteins revealed that they were mainly involved in regulation of transcription (synovial sarcoma X5 protein, metastasis-associated protein 1), cellular reorganization and cytoskeleton (cytokeratins, vimentin, beta actin), cell communication and signal transduction (annexins IV and V, relaxin, APC), and protein synthesis and folding (heat shock protein 60, calreticulin, cathepsin D, RSP4) among others. Preliminary studies demonstrated that the differentially expressed proteins found by 2-D DIGE could be confirmed and validated by immunoblotting and immunohistochemistry analyses in those few cases where antibodies were available. We believe that the incorporation of more samples and new datasets will permit the definition of a collection of proteins with a potential interest as biomarkers for colorectal cancer.

Biopsy↗

PubMed2004

Proteomic analysis of lung biopsies: Differential protein expression profile between peritumoral and tumoral tissue.

The cellular proteome shows a dynamic profile and is subjected to changes in response to various stimuli and disease progression. Lung cancer remains the leading cause of cancer death in industrialized countries. In an attempt to find new disease markers, patients suffering from lung carcinoma have been selected to achieve differential protein expression patterns between normal and neoplasic tissue. After two-dimensional electrophoresis, the spots of interest were digested and identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. This information will provide a more comprehensive understanding of the disease progression and might constitute a method to complement histopathological diagnosis.

Databases, Protein↗

PubMed2004

Identification of cellular proteins modified in response to African swine fever virus infection by proteomics.

Changes in gene expression are produced in cells as a consequence of virus infections. In the present work, we used proteomic technology to globally examine African swine fever virus (ASFV)-infected Vero cells searching for infection-associated proteins in order to determine target proteins for pathogenesis studies. We studied the alterations in cellular protein profile after ASFV infection by two-dimensional electrophoresis, identifying the modified cellular proteins by matrix-assisted laser desorption/ionization peptide mass fingerprinting. A total of twelve different over-expressed cellular proteins were unambiguously identified. The most significant changes were in redox-related proteins, nucleoside diphosphate kinases, heat shock proteins, members of the Ran-Gppnhp-Ranbd1 complex and apolipoproteins. These cellular protein modifications could represent distinct roles during infection related to apoptosis and transcriptional modulation mechanisms. The present study constitutes the first attempt to understand the dynamics of ASFV-host cell interactions by proteomics.

African Swine Fever Virus↗