Molecular & Cellular Proteomics 18.8 1543 * We acknowledge the German Academic Exchange Service (DAAD) for providing scholarships for BD (No. 91566064). The WATERS Synapt G2S mass spectrometer has been bought through an EU grant [EFRE-UHROM 9] made available to MOG.
We have developed a method to determine apparent activation energies of dissociation for ionized protein-protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein-protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ([Formula: see text]) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein-protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.
In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor‐5A (eIF‐5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF‐5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E‐Z‐type HEAT‐like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single‐copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N‐terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F‐type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7255047: DHS (uniprotkb:http://www.ebi.uniprot.org/entry/P49366) enzymaticly reacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0414) with eIF‐5A (uniprotkb:http://www.ebi.uniprot.org/entry/Q710D1) by enzymatic studies (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0415) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7255326: DOHH (uniprotkb:http://www.ebi.uniprot.org/entry/Q8I701) enzymaticly reacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0414) with eIF‐5A (uniprotkb:http://www.ebi.uniprot.org/entry/Q710D1) by enzymatic studies (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0415)
Rheumatoid arthritis is a widespread autoimmune disease. In the murine K/B×N arthritis model, anti-GPI (anti-glucose 6-phosphate isomerase) antibodies lead to the formation of immune complexes. In the course of pathogenesis, these complexes activate the immune system and induce degranulation of mast cells, which are essential in this model of rheumatoid arthritis. A major mediator in mast cell granules is histamine, which is proven to be indispensable for joint inflammation in K/B×N mice. Histamine is known to bind to four different receptors (HR1-4), which have different expression profiles and exert a variety of different functions, including activation of the immune system. To analyze the contribution of the different histamine receptors, we employed histamine receptor antagonists (cetirizine, ranitidine, thioperamide and clozapine) blocking the receptors in C57BL/6 mice. Arthritis was induced via K/B×N serum injection. The results demonstrated that mice treated with all four histamine receptor antagonists simultaneously showed no arthritic symptoms, while positive control mice injected with K/B×N serum and vehicle suffered from severe symptoms. When antagonists specific for HR1-4 were applied individually, only the HR4 antagonist clozapine could protect mice from arthritis, reflecting its expression and functionality in the immune system.
It is well established that arthritis depresses locomotion in humans as well as in animal disease models. The K/BxN mouse model resembles rheumatoid arthritis and is widely used for research. Here, we investigate the behavioral alterations of arthritic K/BxN mice during arthritis development with respect to horizontal locomotion. Locomotor activity measurements and the methodology of ankle thickness measurements are compared to demonstrate the feasibility of motion tracking in the K/BxN mouse model. Arthritic K/BxN mice show significantly decreased locomotion compared to their non-arthritis K/BxN littermates. We found an indirect correlation of ankle thickness and locomotor activity. However, both parameters are only partially interdependent resulting in temporal displacement of maximal ankle swelling and maximal depression of locomotion by 1 week. Assessing the impaired movement as a behavioral test appears to be a valuable multifactorial parameter for the evaluation of arthritis in the K/BxN mouse model and provides additional information on disease progression and severity.
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