The mapping of linear B-cell epitope regions in desmoglein 1 and 3 proteins : Recognition of immobilized peptides by pemphigus patients’ serum autoantibodies

Desmosomal transmembrane glycoproteins desmoglein 1and desmoglein 3 are targets of life-threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus(PF). In these diseases pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell-cell adhesion, and clinically, to the presence of blisters and erosions. Identification, characterization and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding theimmunopathology of the disease and also in the design of novel diagnostics. Here we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins extracellular parts recognized by IgG-type serum autoantibodies of patients with PV andPF. In our study overlapping pentadecapeptides were synthesized on hydroxypropylmethacrylate pins based on the results of in silicopredictions. To detect the interaction between theserum autoantibodies and the immobilized synthetic peptides, modified ELISA (Enzyme Linked Immunosorbent Assay) was performed with pin-attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and four possible epitope regions (aa64-78, aa330-344, aa375-399, aa446-460) within the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in modified ELISAs

Poli(ADP-ribóz) polimeráz gátló vegyületek hatásmechanizmusának vizsgálata állatkísérletes és rheologiai modelleken = Evaluation of mechanism of action of poly(ADP-ribose) polymerase inhibitor compounds in animal and in rheological models

Kutatásaink során kísérletes szívelégtelenség modellekben vizsgálatuk a poli(ADP-ribóz) polimeráz (PARP) gátlók hatását. Vizsgálatainkkal igazoltuk, hogy PARP-gátlók mérséklik a postinfarctusos remodelinget és védenek a szívelégtelenség kialakulásával szemben. Elsőként igazoltuk, hogy a PARP-gátlók jelentős hatással bírnak a szívelégtelenség pathomechanizmusában alapvető szerepet játszó jelátviteli utakra is. PARP-gátlás fokozta a túlélést segítő, ugyanakkor csökkentette bizonyos maladaptív jelátviteli utak aktivitását. Mindezek következtében a PARP-gátlóval kezelt szívek mind funkcionálisan, mind struktúrálisan lényegesen intaktabbak voltak, mint a kezeletlen szívek. Ráadásul az ACE-gátló enalaprillal összehasonítva a PARP-gátló kezelés hatékonyabbnak bizonyult a myocardialis remodeling kivédésében postinfarktusos szívelégtelenség modellben. Fiatal spontán hipertenzív patkányokban a PARP-gátlóknak a szívizom hypertrophia kialakulásával szembeni védő hatását igazoltuk. Ezen eredményeink publikálásra kerültek már, illetve egyesek még publikálás alatt állnak. Emellett idős spontán hipertenzív patkányokban a pangásos szívelégtelenség kialakulásával szemben is kifejezett védő hatást mutattak a PARP-gátlók, az állatok túlélését is javította a kezelés. Ezen adatain még részben feldolgozásra várnak. Doxorubicin kezelés által kiváltott cardiomyopathia modellben végzett vizsgálatunkból származó minták még feldolgozás alatt állnak. | The effect of poly(ADP-ribose) polymerase (PARP) inhibitors was studied in various experimental heart failure models. We have demonstrated that PARP-inhibitors can moderate the postinfarction myocardial remodeling and can protect against the development of heart failure. We have firstly proved that PARP-inhibitors have a significant effect on signal transduction pathways which play a central role in the development of heart failure. PARP-inhibition activated the prosurvival signal transduction pathways and blocked the activity of several maladaptive signal transduction pathways. Due to these effects, hearts treated with PARP-inhibitors showed better functional and structural features compared to untreated hearts. In addition, PARP-inhibition was more effective against the development of myocardial remodeling in our postinfarction heart failure model compared to ACE-inhibition. In young spontaneous hypertensive rats the protective effect of PARP-inhibitors against the development of myocardial hypertrophy was demonstrated. These results were already published and several data are under publication. Moreover, in adult spontaneous hypertensive rats PARP-inhibition protects against the transition from hypertrophic cardiomyopathy to decompensated heart failure. These results will be published shortly. Finally, tissue samples derived from a toxic (doxorubicin-induced) cardiomyopathy model are yet under measurements

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins

The Heat Shock Factor A4A confers salt tolerance and is regulated by oxidative stress and the Mitogen-Activated Protein kinases, MPK3 and MPK6

Heat-shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis. Estradiol-induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays HSFA4A shows homomeric interaction which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, Ser309 being the major phosphorylation site. Activation of the MPK3 and MPK6 MAPK pathway led to the transcriptional activation of the heat-shock protein gene HSP17.6A. In agreement that mutation of Ser309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HSP17.6A. These data suggest that HSFA4A is a substrate of the MPK3/6 signalling and it regulates stress responses in Arabidopsis

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels