Éva Csősz

Functional significance of five noncanonical Ca2+‐binding sites of human transglutaminase 2 characterized by site‐directed mutagenesis

The multifunctional tissue transglutaminase 2 (TG2) has a four‐domain structure with several Ca2+‐regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca2+‐binding form of the human enzyme is not known, and its Ca2+‐binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca2+‐binding residues of epidermal transglutaminase and factor XIIIa (S1–S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody‐binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium‐45 equilibrium dialysis and isothermal calorimetric titration showed that both wild‐type and active site‐deleted enzymes (C277S) bind six Ca2+. Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+‐binding site, and mutation of one site resulted in the loss of more than one bound Ca2+, suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild‐type enzyme, the GTPase activities of the mutants were inhibited by Ca2+, except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca2+‐binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients.

Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy

Diabetic retinopathy is the leading cause of new cases of legal blindness among adults in the developed countries. Approximately 40% of all people with diabetes have diabetic retinopathy and 5% of these have sight-threatening form. As the advanced stage, where there is a high risk for vision loss, can develop without any serious symptoms, sometimes it is hard to detect it. A non invasive method to detect biomarkers characteristic for diabetic retinopathy from the tear fluid was developed. Tear samples from diabetic patients with no retinopathy, non proliferative and proliferative stages of diabetic retinopathy were analyzed and the protein content of each sample was compared to the protein content of tear pool from healthy volunteers. The samples were labeled with iTRAQ fourplex labels and were analyzed with nanoHPLC coupled ESI-MS/MS mass spectrometry. The lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and immunoglobulin lambda chain were identified as possible biomarker candidates with significantly higher relative levels in the tear of patients with diabetic retinopathy.

Transdab wiki: The interactive transglutaminase substrate database on web 2.0 surface

TRANSDAB wiki is a database of transglutaminase substrate proteins. This wiki is designed to provide quality content of all the details (including synonyms, structures, references) about transglutaminase substrate proteins and interaction partners. Currently TRANSDAB contains 243 articles about substrate proteins for 6 transglutaminase types in a user-friendly, editable format. Our aim was to collect structural information about substrate proteins and this information is provided in form of images, videos and links. The scientific community is invited to edit the database and besides providing up-to-date information, this wiki should serve as a platform for valuable discussions.

Quantitative body fluid proteomics in medicine - A focus on minimal invasiveness

Identification of new biomarkers specific for various pathological conditions is an important field in medical sciences. Body fluids have emerging potential in biomarker studies especially those which are continuously available and can be collected by non-invasive means. Changes in the protein composition of body fluids such as tears, saliva, sweat, etc. may provide information on both local and systemic conditions of medical relevance. In this review, our aim is to discuss the quantitative proteomics techniques used in biomarker studies, and to present advances in quantitative body fluid proteomics of non-invasively collectable body fluids with relevance to biomarker identification. The advantages and limitations of the widely used quantitative proteomics techniques are also presented. Based on the reviewed literature, we suggest an ideal pipeline for body fluid analyses aiming at biomarkers discoveries: starting from identification of biomarker candidates by shotgun quantitative proteomics or protein arrays, through verification of potential biomarkers by targeted mass spectrometry, to the antibody-based validation of biomarkers. The importance of body fluids as a rich source of biomarkers is discussed. SIGNIFICANCE Quantitative proteomics is a challenging part of proteomics applications. The body fluids collected by non-invasive means have high relevance in medicine; they are good sources for biomarkers used in establishing the diagnosis, follow up of disease progression and predicting high risk groups. The review presents the most widely used quantitative proteomics techniques in body fluid analysis and lists the potential biomarkers identified in tears, saliva, sweat, nasal mucus and urine for local and systemic diseases.

Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries.

In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 μg·kg⁻¹ daily, for 2 weeks. Even at the highest concentration--a concentration highly toxic in vitro for all affected molds used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy.

Changes in the chemical barrier composition of tears in Alzheimer's disease reveal potential tear diagnostic biomarkers

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, with increasing prevalence affecting millions of people worldwide. Currently, only autopsy is able to confirm the diagnosis with a 100% certainty, therefore, biomarkers from body fluids obtained by non-invasive means provide an attractive alternative for the diagnosis of Alzheimer`s disease. Global changes of the protein profile were examined by quantitative proteomics; firstly, electrophoresis and LC-MS/MS were used, thereafter, SRM-based targeted proteomics method was developed and applied to examine quantitative changes of tear proteins. Alterations in the tear flow rate, total tear protein concentration and composition of the chemical barrier specific to AD were demonstrated, and the combination of lipocalin-1, dermcidin, lysozyme-C and lacritin was shown to be a potential biomarker, with an 81% sensitivity and 77% specificity.

Proteomics investigation of OSCC-specific salivary biomarkers in a Hungarian population highlights the importance of identification of population-tailored biomarkers

Oral squamous cell carcinoma (OSCC) accounting for about 90% of malignant oral lesions is the 6th most common malignancy worldwide. Diagnostic delay may contribute to dismal survival rate therefore, there is a need for developing specific and sensitive biomarkers to improve early detection. Hungarian population occupies the top places of statistics regarding OSCC incidence and mortality figures therefore, we aimed at finding potential salivary protein biomarkers suitable for the Hungarian population. In this study we investigated 14 proteins which were previously reported as significantly elevated in saliva of patients with OSCC. In case of IL-1α, IL-1β, IL-6, IL-8, TNF-α and VEGF a Luminex-based multiplex kit was utilized and the salivary concentrations were determined. In case of catalase, profilin-1, S100A9, CD59, galectin-3-bindig protein, CD44, thioredoxin and keratin-19, SRM-based targeted proteomic method was developed and the relative amount of the proteins was determined in the saliva of patients with OSCC and controls. After several rounds of optimization and using stable isotope-containing peptides, we developed an SRM-based method for rapid salivary protein detection. The validation of the selected potential biomarkers by ELISA revealed salivary protein S100A9 and IL-6 as useful protein biomarkers for OSCC detection improving the diagnostic accuracy for OSCC in the Hungarian population.A noninvasive diagnostic method to detect biomarkers useful for the early diagnosis of OSCC was developed. This can be an attractive strategy in screening saliva samples collected in a nation-wide multi-centric study in order to decrease morbidity, mortality, to enhance survival rate and to improve quality of life. The heterogeneity of protein biomarkers found in different ethnic groups presented in the literature highlights the importance of identification of population-tailored protein biomarkers.

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.

Interactions of retinoids with the ABC transporters P-glycoprotein and Breast Cancer Resistance Protein

Retinoids – derivatives of vitamin A – are important cell permeant signaling molecules that regulate gene expression through activation of nuclear receptors. P-glycoprotein (Pgp) and ABCG2 are plasma membrane efflux transporters affecting the tissue distribution of numerous structurally unrelated lipophilic compounds. In the present work we aimed to study the interaction of the above ABC transporters with retinoid derivatives. We have found that 13-cis-retinoic acid, retinol and retinyl-acetate inhibited the Pgp and ABCG2 mediated substrate transport as well as the substrate stimulated ATPase activity of these transporters. Interestingly, 9-cis-retinoic acid and ATRA (all-trans retinoic acid), both are stereoisomers of 13-cis-retinoic acid, did not have any effect on the transporters’ activity. Our fluorescence anisotropy measurements revealed that 13-cis-retinoic acid, retinol and retinyl-acetate selectively increase the viscosity and packing density of the membrane. Thus, the mixed-type inhibition of both transporters by retinol and ABCG2 by 13-cis-retinoic acid may be the collective result of direct interactions of these retinoids with the substrate binding site(s) and of indirect interactions mediated by their membrane rigidifying effects.