Mass spectrometric detection of affinity purified crosslinked presented

Hurst, Gregory B.; Lankford, Trish K.; Kennel, Stephen J.

doi:10.1016/j.jasms.2004.02.008

Cited by 61 publications

(20 citation statements)

References 29 publications

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“…The interacting complex is then isolated and the disulfide bond subsequently reduced. Upon reduction of the disulfide bond, PCMT is released and biotin is “transferred” onto the methyltransferase substrate (Label Transfer Method) [21] (Fig. 5 panel A).…”

Section: Resultsmentioning

confidence: 99%

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

et al. 2008

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BackgroundNatural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).Methodology/Principal FindingsEndothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H2O2; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H2O2 concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H2O2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.Conclusion/SignificanceApoptosis can be modulated by “on/off” switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

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Section: Resultsmentioning

confidence: 99%

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

et al. 2008

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“…To more directly identify the binding domain, we have used a photoactive cross-linking approach with sulfosuccinimidyl-2-[6-(biotinamido)-2-( p -azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) [18]. This cross-linker has 4 functional groups: a NHS-ester group, a UV-activatable aryl azide group, a cleavable disulfide bond, and a biotin group (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

“…The use of photoactive cross-linkers, followed by identification of cross-linked regions by MS analysis has proven useful in complex interactions, especially where tertiary conformational factors play a role [18], [26], [27]. Using that method, we identified two P-domain sequences located, respectively, in positions 196–211 and 262–275 of the CRT P-domain.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

et al. 2010

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BackgroundThe rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRβ chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of - RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT.Principal FindingsSurface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu217 and Glu223 - and to a lesser extent residue Asp220 - in cell-free SPR-based binding and signal transduction assays.SignificanceWe have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

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“…Affinity purification strategies have been employed to aid the purification of crosslinked protein complexes 13, 18, 19 . Crosslinkers with defined isotope tags have been used to distinguish crosslinker-modified peptides from unmodified peptides as well as for quantitative analysis of crosslinked peptides 15, 16, 20, 21 .…”

Section: Introductionmentioning

confidence: 99%

Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy

et al. 2008

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Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying crosslinked peptides from single LC/MS/MS datasets, without using specialized crosslinkers or custom-written software. To achieve this, each peptide in the pair of crosslinked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of crosslinked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich datasets for crosslinked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native crosslinks in proteins. The pipeline was validated by characterizing the known interactions in chemically crosslinked CYP2E1-b5 complex. Utility of this method in identifying native crosslinks was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.

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Mass spectrometric detection of affinity purified crosslinked presented

Cited by 61 publications

References 29 publications

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy

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