Proteomic analysis identifies differentially expressed proteins in AML1/ETO acute myeloid leukemia cells treated with DNMT inhibitors azacitidine and decitabine.

Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias. To further characterize different mechanisms between these two agents, cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis. Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS. Cyclophilin A, Catalase, Nucleophosmin and PCNA were decreased exclusively by azacitidine, TCP1 and hnRNP A2/B1 by both drugs; alpha-Enolase and Peroxiredoxin-1 by decitabine. Interestingly, the expression of the proinflammatory protein Cyclophilin A, also suggested as marker of cell necrosis, was stimulated by decitabine. Finally, a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors.

Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid.

Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.

In vitro transcription and translation coupled to two-dimensional electrophoresis for bacterial proteome analysis.

The most popular approach for proteomic analysis is based on the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. Although very effective, the method suffers from a number of limitations, the most serious one being the necessity of expensive and sophisticated instrumentation to be operated by skilled personnel. Here, we propose an alternative approach, which is particularly useful when one is interested to establish if a subset of proteins is present in a complex protein mixture derived from a sequenced organism. The method is based on amplification of the genes whose products are under investigation. The amplified genes are used in transcription and translation reactions and the derived radio-labeled proteins are separated by 2DE. The gel is autoradiographed and the autoradiograph is superimposed on the 2D gel (sample gel) from which the protein mixture from the organism has been separated. The matching between the autoradiographic spots and the protein spots of the sample gel allows immediate protein identification.

Comparative proteomic analysis of chronic myelogenous leukemia cells: inside the mechanism of imatinib resistance.

Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.

Outer membrane vesicles from group B Neisseria meningitidis delta gna33 mutant: proteomic and immunological comparison with detergent-derived outer membrane vesicles.

We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) spontaneously released into culture supernatant by MenB delta gna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m-OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane composition and organization, and to design new efficacious vaccine formulations.

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

Identification of new potential vaccine candidates against Chlamydia pneumoniae by multiple screenings.

Chlamydia are intracellular bacteria associated to serious human disease. A vaccine has proved difficult to obtain so far, and current opinions agree that multi-antigen combinations may be required to induce optimal protective responses. In order to identify new potential vaccine candidates, we recently screened the Chlamydia pneumoniae (Cpn) genome and described 53 recombinant proteins which elicited antibodies binding to purified Cpn cells. We now report that six proteins in this group can also induce in vitro neutralizing antibodies. Antibody specificity for the corresponding antigens was assessed by immunoblot analysis of 2DE Cpn protein maps. Furthermore, four of the six in vitro neutralizing antigens (Pmp2, Pmp10, OmpH-like and enolase) could inhibit Cpn dissemination in a hamster model. The results show that these Cpn proteins are immunoaccessible in infectious EBs, and recommend further investigation on their value as vaccine components.

GNA33 of Neisseria meningitidis is a lipoprotein required for cell separation, membrane architecture, and virulence.

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.