4 research outputs found
Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1
Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively
Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β<sub>2</sub> on Monocytes via NADPH Oxidase
Physiological
stimuli such as thrombin, or pathological stimuli
such as lysophosphatidic acid (LPA), activate platelets. The activated
platelets bind to monocytes through P-selectin–PSGL-1 interactions
but also release the contents of their granules, commonly called “platelet
releasate”. It is known that monocytes in contact with platelet
releasate produce reactive oxygen species (ROS). Reversible cysteine
oxidation by ROS is considered to be a potential regulator of protein
function. In a previous study, we used THP-1 monocytic cells exposed
to LPA- or thrombin-induced platelet releasate and a modified biotin
switch assay to unravel the biological processes that are influenced
by reversible cysteine oxidation. To gain a better understanding of
the redox regulation of monocytes in atherosclerosis, we have now
altered the modified biotin switch to selectively quantify protein
sulfenic acid, a subpopulation of reversible cysteine oxidation. Using
arsenite as reducing agent in the modified biotin switch assay, we
were able to quantify 1161 proteins, in which more than 100 sulfenic
acid sites were identified. Bioinformatics analysis of the quantified
sulfenic acid sites highlighted the relevant, previously missed biological
process of monocyte transendothelial migration, which included integrin
β<sub>2</sub>. Flow cytometry validated the activation of LFA-1
(α<sub>L</sub>β<sub>2</sub>) and Mac-1 (α<sub>M</sub>β<sub>2</sub>), two subfamilies of integrin β<sub>2</sub> complexes, on human primary monocytes following platelet releasate
treatment. The activation of LFA-1 was mediated by ROS from NADPH
oxidase (NOX) activation. Production of ROS and activation of LFA-1
in human primary monocytes were independent of P-selectin–PSGL-1
interaction. Our results proved the modified biotin switch assay to
be a powerful tool with the ability to reveal new regulatory mechanisms
and identify new therapeutic targets