[The Role of Calcium in the Conformational Changes of the Recombinant S100A8/S100A9].

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.

Imaging mass spectrometry for assessing temporal proteomics: analysis of calprotectin in Acinetobacter baumannii pulmonary infection.

Imaging MS is routinely used to show spatial localization of proteins within a tissue sample and can also be employed to study temporal protein dynamics. The antimicrobial S100 protein calprotectin, a heterodimer of subunits S100A8 and S100A9, is an abundant cytosolic component of neutrophils. Using imaging MS, calprotectin can be detected as a marker of the inflammatory response to bacterial challenge. In a murine model of Acinetobacter baumannii pneumonia, protein images of S100A8 and S100A9 collected at different time points throughout infection aid in visualization of the innate immune response to this pathogen. Calprotectin is detectable within 6 h of infection as immune cells respond to the invading pathogen. As the bacterial burden decreases, signals from the inflammatory proteins decrease. Calprotectin is no longer detectable 96-144 h post infection, correlating to a lack of detectable bacterial burden in lungs. These experiments provide a label-free, multiplexed approach to study host response to a bacterial threat and eventual clearance of the pathogen over time.

S100A8/A9 amyloidosis in the ageing prostate: relating ex vivo and in vitro studies.

The family of S100 proteins encompasses more than 20 members characterized by remarkable conformational and functional diversity. S100 proteins act as central regulators of various cellular processes, including cell survival, proliferation, differentiation, and motility. Many S100 proteins are implicated in various types of cancer as well as neurodegenerative, inflammatory, and autoimmune diseases. Recently, we have found that S100A8/A9 proteins are involved in amyloidogenic process in the ageing prostate, contributing to the formation of calcified corpora amylacea (CA) inclusions, which commonly accompany age-dependent prostate tissue remodelling and cancer. Amyloid formation by S100A8/A9 proteins can also be modelled in vitro. Amyloid assembly of S100A8/A9 proteins into oligomeric and fibrillar complexes is modulated by metal ions such as calcium and zinc. Here, we provide insights into the extraction procedures and review the common structural features of ex vivo and in vitro S100A8/A9 amyloids, showing that they share the same generic origin.

Proteome analysis of human amnion and amniotic fluid by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Proteome analysis by 2-DE and PMF by MALDI-TOF MS was performed on human amnion and amniotic fluid at term. Ninety-two soluble and nineteen membrane proteins were identified from amnion. Thirty-five proteins were identified from amniotic fluid. Calgranulin A and B were found in all patients infected with Ureaplasma urealyticum, but not in any of the patients without infection, indicating that they are potential markers of intrauterine infection. Identity of calgranulin A and B was confirmed by MALDI-TOF/TOF MS. This study represents the first extensive analysis of the human amnion and amniotic fluid proteome at term and demonstrates that 2-DE and MALDI-TOF MS is a useful tool for identifying clinically significant biomarkers of problematic pregnancies.

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation.

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.