Biomedical subjects

Daniel Boismenu

Publications and source records attributed to Daniel Boismenu.

9 recordsLinked to original sources

PubMed2007

Proteomic analysis of tyrosine phosphorylation during human liver transplantation.

BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

Journal Article↗

PubMed2006

Proteomic analysis of ischemia-reperfusion injury upon human liver transplantation reveals the protective role of IQGAP1.

Ischemia-reperfusion injury (IRI) represents a major determinant of liver transplantation. IRI-induced graft dysfunction is related to biliary damage, partly due to a loss of bile canaliculi (BC) integrity associated with a dramatic remodeling of actin cytoskeleton. However, the molecular mechanisms associated with these events remain poorly characterized. Using liver biopsies collected during the early phases of organ procurement (ischemia) and transplantation (reperfusion), we characterized the global patterns of expression and phosphorylation of cytoskeleton-related proteins during hepatic IRI. This targeted functional proteomic approach, which combined protein expression pattern profiling and phosphoprotein enrichment followed by mass spectrometry analysis, allowed us to identify IQGAP1, a Cdc42/Rac1 effector, as a potential regulator of actin cytoskeleton remodeling and maintenance of BC integrity. Cell fractionation and immunohistochemistry revealed that IQGAP1 expression and localization were affected upon IRI and related to actin reorganization. Furthermore using an IRI model in human hepatoma cells, we demonstrated that IQGAP1 silencing decreased the basal level of actin polymerization at BC periphery, reflecting a defect in BC structure coincident with reduced cellular resistance to IRI. In summary, this study uncovered new mechanistic insights into the global regulation of IRI-induced cytoskeleton remodeling and led to the identification of IQGAP1 as a regulator of BC structure. IQGAP1 therefore represents a potential target for the design of new organ preservation strategies to improve transplantation outcome.

Actins↗

PubMed2005

Characterization of an RNA granule from developing brain.

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.

Actins↗

PubMed2005

Multicomponent internal recalibration of an LC-FTICR-MS analysis employing a partially characterized complex peptide mixture: systematic and random errors.

In high-throughput proteomics, a promising current approach is the use of liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) of tryptic peptides from complex mixtures of proteins. To apply this method, it is necessary to account for any systematic measurement error, and it is useful to have an estimate of the random error expected in the measured masses. Here, we analyze by LC-FTICR-MS a complex mixture of peptides derived from a sample previously characterized by LC-QTOF-MS. Application of a Bayesian probability model of the data and partial knowledge of the composition of the sample suffice to estimate both the systematic and random errors in measured masses.

Amino Acid Sequence↗

PubMed2005

Increasing peptide identification in tandem mass spectrometry through automatic function switching optimization.

Comprehensive proteomic studies that employ MS directed peptide sequencing are limited by optimal peptide separation and MS and tandem MS data acquisition routines. To identify the optimal parameters for data acquisition, we developed a system that models the automatic function switching behavior of a mass spectrometer using an MS-only dataset. Simulations were conducted to characterize the number and the quality of simulated fragmentation as a function of the data acquisition routines and used to construct operating curves defining tandem mass spectra quality and the number of peptides fragmented. Results demonstrated that one could optimize for quality or quantity, with the number of peptides fragmented decreasing as quality increased. The predicted optimal operating curve indicated that significant improvements can be realized by selecting the appropriate data acquisition parameters. The simulation results were confirmed experimentally by testing 10 LC MS/MS data acquisition parameter sets on an LC-Q-TOF-MS. Database matching of the experimental fragmentation returned peptide scores consistent with the predictions of the model. The results of the simulations of mass spectrometer data acquisition routines reveal an inverse relationship between the quality and the quantity of peptide identifications and predict an optimal operating curve that can be used to select an optimal data acquisition parameter for a given (or any) sample.

Algorithms↗

PubMed2004

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.

Animals↗

PubMed2002

The model B6(dom1) minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene.

The B6(dom1) minor histocompatibility antigen (MiHA) is a model antigen, since it is both the epitome of an immunodominant epitope and an ideal target for adoptive cancer immunotherapy. Based on DNA sequencing and MS/MS analyses, we report that B6(dom1) corresponds to amino acids 770-778 (KAPDNRETL) of a protein we propose to call SIMP (source of immunodominant MHC-associated peptides) that is encoded by a mouse homolog of the yeast STT3gene. STT3, a member of the oligosaccharyltransferase complex, is essential for cell proliferation. Phenotypic and genotypic analyses among eight strains of mice revealed a precise correlation between susceptibility or resistance to B6(dom1)-specific cytotoxic T lymphocytes (CTLs) and the presence of a Glu vs Asp amino acid at position 776 of the SIMP protein, respectively. Strikingly, while the difference in the amino acid sequence 770-778 encoded by the two SIMP alleles represents a very conservative substitution, these allelic peptides were not crossreactive at the CTL level, and both peptides were immunodominant when presented to mice homozygous for the opposite allele. In addition, we have cloned a human ortholog of SIMP whose predicted protein shares 97% amino acid identity with mouse SIMP. These results strengthen the concept that MHC class-I-associated MiHAs originate as a consequence of rare polymorphisms among highly conserved genes. Furthermore, the notion that a peptide differing from a self analog by a single methylene group can be immunodominant has implications regarding our understanding of the mechanisms of immunodominance.

Amino Acid Sequence↗

PubMed2002

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics.

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, gamma-ear-containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins.

Adaptor Protein Complex 1↗

PubMed2002

Concentration-effect relation of succinylcholine chloride during propofol anesthesia.

BACKGROUND: The pharmacokinetics and pharmacodynamics of succinylcholine were studied simultaneously in anesthetized patients to understand why the drug has a rapid onset and short duration of action. A quantitative model describing the concentration-effect relation of succinylcholine was proposed. The correlation between hydrolysis in plasma and elimination was also examined. METHODS: Before induction of anesthesia, blood was drawn for analysis in seven adults. Anesthesia was induced with propofol and remifentanil. Single twitch stimulation was applied at the ulnar nerve every 10 s, and the force of contraction of the adductor pollicis was measured. Arterial blood was drawn frequently after succinylcholine injection to characterize the front-end kinetics. Plasma concentrations were measured by mass spectrometry, and pharmacokinetic parameters were derived using compartmental and noncompartmental approaches. Pharmacokinetic-pharmacodynamic relations were estimated. RESULTS: The mean degradation rate constant in plasma (1.07 +/- 0.49 min(-1)) was not different from the elimination rate constant (0.97 +/- 0.30 min(-1)), and an excellent correlation (r2 = 0.94) was observed. Total body clearance derived using noncompartmental (37 +/- 7 ml x min(-1) x kg(-1)) and compartmental (37 +/- 9 ml x min(-1) x kg(-1)) approaches were similar. The plasma-effect compartment equilibration rate constant (k(eo)) was 0.058 +/- 0.026 min(-1), and the effect compartment concentration at 50% block was 734 +/- 211 ng/ml. CONCLUSION: Succinylcholine is a low-potency drug with a very fast clearance that equilibrates relatively slowly with the effect compartment. Its disappearance is greatly accountable by a rapid hydrolysis in plasma.

Adult↗