Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis (Arabidopsis thaliana) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S::AtSCS-A-c-myc or 35S::AtSCS-B-c-myc in the scs-1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins

3. Losartan and Enalapril in the Treatment of Essentail Hypertension — Effect on LeftVentricular Mass and Function

Wstęp: Celem badania była ocena wpływu losartanu i enalaprylu na ciśnienie tętnicze, masę lewej komory i wybrane parametry jej funkcji u pacjentów z nadciśnieniem tętniczym. Materiał i metody: Badaniami objęto 61 pacjentów (43 mężczyzn i 18 kobiet) w wieku 37–70 lat (średnio 56,8 ± 8 lat). Losartan podawano 31 pacjentom w dawce 50 mg/d., enalapryl — 30 chorym w dawce 10–20 mg/d. przez 12 miesięcy. U 17 pacjentów leczonych losartanem i u 18 leczonych enalaprylem występował przerost lewej komory. Pomiary wartoś- ci ciśnienia tętniczego i badania echokardiograficzne wykonywano przed rozpoczęciem leczenia oraz po 1, 3, 9 i 12 miesiącach terapii. Wyniki: W początkowym okresie terapii większy efekt hipotensyjny obserwowano w grupie pacjentów bez przerostu lewej komory. Po 3 miesiącach leczenia losartanem i enalaprylem efekt hipotensyjny był podobny u pacjentów z przerostem lewej komory i bez niego. Po 3–6 miesiącach terapii losartanem i enalaprylem obniżył się wskaźnik masy lewej komory, a frakcja wyrzutowa się zwiększyła. Stosunek wczesnego do późnego napełniania lewej komory (EV/AV) wzrósł natomiast po 1–3 miesiącach terapii.Analiza zmian parametrów echokardiograficznych wykazała, że współczynnik masy lewej komory zmalał jedynie w grupie pacjentów z przerostem lewej komory. Wnioski: Losartan i enalapryl wykazują podobne działanie hipotensyjne oraz podobny wpływ na masę i funkcję lewej komory u osób z nadciśnieniem tętniczym.Background: The aim of the study was to assess the effect of losartan and enalapril on arterial blood pressure and left ventricular mass and selected parameters of its function in patients with hypertension. Material and methods Examinations were performed in 61 subjects (43 men and 18 women) aged 37–70 years (mean 56,8 ± 8). Losartan was applied in 31 subjects in a dose 50 mg/24 h, while enalapril in 30 subjects in a dose 10–20 mg/24 h for 12 months. 17 subjects on losartan and 18 on enalapril had left ventricular hypertrophy. Arterial pressure measurements and echocardiography were performed before and after 1, 3, 9 and 12 months of treatment. Results: In the initial period of the therapy greater hypotensive effect was observed in the group of patients without ventricular hypertrophy; after 3 months losartan and enalapril hypotensive effect was similar in subjects with and without ventricular hypertrophy. Left ventricular mass index decreased, ejection fraction increased after 3–6 months of losartan and enalapril therapy, while early to atrial peak flow velocity ratio (E/A) increased after 1–3 months of the therapy. Analysis of the changes of echocardiographic parameters values showed that left ventricular mass index decreased only in the group of patients with left ventricular hypertrophy. Conclusion: Losartan and enalapril demonstrate similar hypotensive effect and similar effect on left ventricular mass and function in subjects with essential hypertension

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quaternary structure formation and activity.

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function

Regulation of ABA-Non-Activated SNF1-Related Protein Kinase 2 Signaling Pathways by Phosphatidic Acid

Phosphatidic acid (PA) is involved in the regulation of plant growth and development, as well as responses to various environmental stimuli. Several PA targets in plant cells were identified, including two SNF1-related protein kinases 2 (SnRK2s), SnRK2.10 and SnRK2.4, which are not activated by abscisic acid (ABA). Here, we investigated the effects of PA on various elements of ABA-non-activated SnRK2 signaling. PA 16:0/18:1 was found to modulate the SnRK2 structure and the phosphorylation of some SnRK2 targets. Conversely, phosphorylation by the ABA-non-activated SnRK2s, of one of such targets, dehydrin Early Responsive to Dehydration 14 (ERD14), affects its interaction with PA and subcellular localization. Moreover, PA 16:0/18:1 modulates the activity and/or localization of negative regulators of the ABA-non-activated SnRK2s, not only of the ABA insensitive 1 (ABI1) phosphatase, which was identified earlier, but also of another protein phosphatase 2C, PP2CA. The activity of both phosphatases was inhibited by about 50% in the presence of 50 μM PA. PA 16:0/18:1 also impacts the phosphorylation and subcellular localization of SnRK2-interacting calcium sensor, known to inhibit SnRK2 activity in a calcium-dependent manner. Thus, PA was found to regulate ABA-non-activated SnRK2 signaling at several levels: the activity, phosphorylation status and/or localization of SnRK2 cellular partners

Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quarternary structure formation and activity

Arabidopsis thaliana AtNUDT7, a homodimeric Nudix hydrolase active on ADP-ribose and NADH, exerts negative control on the major signaling complex involved in plant defense activation and programmed cell death. The structural and functional consequences of altering several amino-acid residues of the AtNUDT7 protein have been examined by site-directed mutagenesis, far-UV circular dichroism (CD), attenuated total reflection-Fourier transform infrared (ATR-FTIR) and photon correlation (PCS) spectroscopy, biochemical analysis and protein-protein interaction studies. Alanine substitutions of F73 and V168 disallowed dimer formation. Both the F73A- and V168A-mutated proteins displayed no observable enzymatic activity. Alanine substitution of the V69 residue did not significantly alter the enzyme activity and had no influence on dimer arrangement. The non-conserved V26 residue, used as a negative control, did not contribute to the enzyme quaternary structure or activity. Detailed biophysical characterization of the wild-type and mutant proteins indicates that the mutations do not considerably alter the secondary structure of the enzyme but they affect dimer assembly. In addition, mutating residues V69, F73 and V168 disrupted the binding of AtNUDT7 to the regulatory 14.3.3 protein. These are the first studies of the structure-function relationship of AtNUDT7, a Nudix hydrolase of important regulatory function

Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1 and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy