Jane E. Findlay

The UV-B photoreceptor UVR8 promotes photosynthetic efficiency in Arabidopsis thaliana exposed to elevated levels of UV-B

The UV-B photoreceptor UVR8 regulates expression of genes in response to UV-B, some encoding chloroplast proteins, but the importance of UVR8 in maintaining photosynthetic competence is unknown. The maximum quantum yield of PSII (Fv/Fm) and the operating efficiency of PSII (ΦPSII) were measured in wild-type and uvr8 mutant Arabidopsis thaliana. The importance of specific UVR8-regulated genes in maintaining photosynthetic competence was examined using mutants. Both Fv/Fm and ΦPSII decreased when plants were exposed to elevated UV-B, in general more so in uvr8 mutant plants than wild-type. UV-B increased the level of psbD-BLRP (blue light responsive promoter) transcripts, encoding the PSII D2 protein. This increase was mediated by the UVR8-regulated chloroplast RNA polymerase sigma factor SIG5, but SIG5 was not required to maintain photosynthetic efficiency at elevated UV-B. Levels of the D1 protein of PSII decreased markedly when plants were exposed to elevated UV-B, but there was no significant difference between wild-type and uvr8 under conditions where the mutant showed increased photoinhibition. The results show that UVR8 promotes photosynthetic efficiency at elevated levels of UV-B. Loss of the DI polypeptide is probably important in causing photoinhibition, but does not entirely explain the reduced photosynthetic efficiency of the uvr8 mutant compared to wild-type.

Targeted disruption of the heat shock protein 20–phosphodiesterase 4D (PDE4D) interaction protects against pathological cardiac remodelling in a mouse model of hypertrophy

Phosphorylated heat shock protein 20 (HSP20) is cardioprotective. Using human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and a mouse model of pressure overload mediated hypertrophy, we show that peptide disruption of the HSP20–phosphodiesterase 4D (PDE4D) complex results in attenuation of action potential prolongation and protection against adverse cardiac remodelling. The later was evidenced by improved contractility, decreased heart weight to body weight ratio, and reduced interstitial and perivascular fibrosis. This study demonstrates that disruption of the specific HSP20–PDE4D interaction leads to attenuation of pathological cardiac remodelling.

Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

The cyclic AMP‐specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kinase, and we propose that this mechanism forms a feedback loop that results in the restoration of basal cAMP levels.

Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

ABSTRACT Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells. It forms a complex with both focal adhesion kinase (FAK, also known as PTK2) and Src in SCC cells derived from skin carcinomas induced by administration of the chemical DMBA followed by TPA (the DMBA/TPA model). Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with Src and drives trafficking of Src to autophagic structures that SCC cells use to cope with high levels of active Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, thereby meaning active Src becomes tethered at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

Compaction of a prokaryotic signal-anchor transmembrane domain begins within the ribosome tunnel and is stabilized by SRP during targeting

Cotranslational targeting of membrane proteins is mediated by the universally conserved signal recognition particle (SRP). In eukaryotes, SRP attenuates translation during targeting; however, in prokaryotes, a simplified SRP is believed to carry out targeting during continuing translation. Here, we show a detailed stepwise analysis of the targeting of subunit c of the F(0) component of the bacterial ATP synthase (F(0)c) to the inner membrane. We show that the first transmembrane (TM) signal-anchor domain of F(0)c forms a compacted structure within the distal portion of the ribosome tunnel. This structure is formed just prior to the interaction with SRP. In the absence of SRP this structure is lost as the TM domain exits the tunnel; however in the presence of SRP it is stabilized. Our results suggest differences in early protein folding of substrates for prokaryotic SRP-dependent membrane protein targeting pathways, from that of eukaryotic SRP targeting. These results imply that early TM domain recognition by targeting factors acts to ensure that the efficiency of membrane targeting is maintained.

FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system

Disrupted in schizophrenia 1 (DISC1) is a multi-functional scaffolding protein that has been associated with neuropsychiatric disease. The role of DISC1 is to assemble protein complexes that promote neural development and signaling, hence tight control of the concentration of cellular DISC1 in neurons is vital to brain function. Using structural and biochemical techniques, we show for we believe the first time that not only is DISC1 turnover elicited by the ubiquitin proteasome system (UPS) but that it is orchestrated by the F-Box protein, FBXW7. We present the structure of FBXW7 bound to the DISC1 phosphodegron motif and exploit this information to prove that disruption of the FBXW7-DISC1 complex results in a stabilization of DISC1. This action can counteract DISC1 deficiencies observed in neural progenitor cells derived from induced pluripotent stem cells from schizophrenia patients with a DISC1 frameshift mutation. Thus manipulation of DISC1 levels via the UPS may provide a novel method to explore DISC1 function.

OS 15-08 Ang(1-7) INFLUENCES ET-1 SIGNALING THROUGH MAS: ETBR INTERACTIONS: IMPLICATIONS IN PULMONARY HYPERTENSION.

Objective: Ang(1–7) has been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the importance of ET-1 in PH pathophysiology and endothelial dysfunction, we questioned whether Ang(1–7) influences ET-1 signaling in endothelial cells and whether Ang(1–7) treatment influences the ET-1 system in PH. Design and Method: Human endothelial cells (hEC) were stimulated with ET-1 in absence/presence of Ang(1–7). Mas and ETBR interaction was observed by immunoprecipitation. To characterize physical interactions, we utilized novel technology, employing a library of peptides spanning the MasR sequence, to define sites of ETBR binding. To investigate pathophysiological significance of our findings, we investigated whether Ang(1–7) treatment ameliorates PH. Hypoxia was used to induce PH in mice: normoxic (NV) and hypoxic vehicle (HV), normoxic (NA) and hypoxic PH (HA) treated with Ang(1–7) 30 &mgr;g/kg/day. Results: Ang(1–7) increases ET-1 release (125%) and ETBR protein (50%). ET-1-induced increases in VCAM-1 protein (38%) and TNF&agr; production (30%) were blocked by Ang(1–7). Pro-inflammatory effects were dependent on NO. Ang(1–7) increased NO production (257%) in a Mas and ETBR-dependent manner. Mutagenesis studies identified regions conferring specificity for ETBR binding. Peptide disruptors to prevent Mas/ETBR interaction were used for in vitro validation. We previously demonstrated in hEC that Ang(1–7) stimulates eNOS phosphorylation (180%), an effect inhibited by pre-incubation with peptide disruptors. In HP mice, RVSP (18.7 NV vs. 47.6mmHg HV, p < 0.05) RVH (0.19 NV vs. 0.28 HV, p < 0.01) and ET-1 levels (0.8 NV vs 2.4pg/ml HV, p < 0.05) were increased and blocked by Ang(1–7). Hypercontractility in pulmonary arteries of HV mice was attenuated by Ang(1–7). Conclusions: These findings indicate that vasoprotective effects of Ang(1–7) may be mediated through Mas:ETBR dimerization. In vivo studies support a relationship between Ang(1–7)/Mas and ET-1 systems. In conclusion we have identified a novel link between Ang(1–7) and ET-1 through physical interactions between Mas and ETBR.

JS ISH-ECCR-2 ANG-(1-7) AND ET-1, A NEW PARTNERSHIP.

ACE2 and Ang-1-7 have been shown to protect against pulmonary hypertension (PH). Mechanisms for this remain unclear. Considering the important role of ET-1 in the pathophysiology of PH and endothelial dysfunction, we questioned whether Ang-(1-7) influences ET-1 signaling in endothelial cells and whether Ang-(1-7) treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in the absence/presence of Ang 1-7 and showed that Ang 1-7 increased preproET-1 mRNA levels, ET-1 release, and ETBR protein levels. ET-1 increases in e-selectin mRNA, VCAM-1 protein and TNFα production were blocked by Ang 1-7. Pro-inflammatory effects were dependent on NO production. Ang 1-7 increased NO production in a Mas and ETBR-dependent manner. An interaction between Mas and ETBR was observed by immunoprecipitation. To further characterise a physical interaction between Mas/ETBR, we utilised novel technology, employing a library of overlapping peptides scanning the entirety of the MasR sequence, to define the interaction sites for ETBR binding. By substitution or sequence truncation we identified two distinct regions on the MasR that confer specificity for ETBR binding. Peptides that disrupt each of these regions to prevent Mas/ETBR interaction were developed for in vitro validation. To investigate the pathophysiological significance of our findings, we investigated whether Ang-(1-7) treatment ameliorates PH and whether this is associated with changes in ET-1 status. Hypobaric hypoxia was used to induce PH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with orally active Ang 1-7 30 μg/kg/day for 14 days. In HP mice, RVSP, RVH and ET-1 levels were increased and blocked by Ang 1-7 treatment. Hyper-contractility and endothelial dysfunction in pulmonary arteries of HP mice compared to NC was attenuated by Ang 1-7. These findings indicate that vasoprotective effects of Ang-(1-7) may be mediated through dimerization of MAS:ETBR. In vivo studies support a relationship between the Ang-(1-7)/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang-(1-7) and ET-1 through physical interactions between MAS and ETBR.

10 Sumoylation of essential cardiac signalling proteins: preliminary evidence

SUMOylation is a post-translational modification process involving conjugation of the SUMO protein (small ubiquitin-like modifiers) to lysine residues, which can affect protein structure, function and subcellular location. Previous studies by Hajjar et al .1 have demonstrated SUMOylation of SERCA2a, a calcium transporting ATPase responsible for calcium ion reuptake during cardiac excitation contraction coupling. It has been shown that SUMOylation preserves the ATPase activity and stability of SERCA2a in both mouse and human cells and that levels of SUMO1 and the SUMOylation of SERCA2a itself is greatly reduced in heart failure. Moreover, reconstitution of SUMO1 via adeno-associated-virus-mediated gene delivery has been shown to maintain expression of SERCA2a as well as improving cardiac function in mice with heart failure. Using sequence analysis, we have identified putative SUMOylation sites on other cardiac signalling proteins (beta2 adrenoceptor, ryanodine receptor, cardiac troponin I, and l-type calcium channel) and present preliminary evidence using peptide array that SUMOylation of these sites may occur. As proof of concept we have raised a specific antibody against the SUMOylated form of cTNI and show that this reagent can recognise the SUMOylated form of the protein in cells. We speculate that SUMOylation of cardiac proteins may be utilised as a biomarker of heart disease or provide a platform for the design of novel therapeutic strategies for heart failure. Reference Kho C, Lee A, Jeong D, Oh JG, Chaanine AH, Kizana E, Park WJ, Hajjar RJ. SERCA2a (sarcoplasmic reticulum Ca2+ ATPase) cTNI (cardiac troponin I). Nature 2011;477:601–605

3 Angiotensin 1–7 regulation of endothelin-1 system in pulmonary hypertension

ACE2 and Ang1–7 have been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the important role of ET-1 in PH pathophysiology and endothelial dysfunction, we asked whether Ang1–7 influences ET-1 signalling in PH. Human endothelial cells (HMEC) were stimulated with ET-1 in absence/presence of Ang1–7 and showed that Ang1–7 increased ET-1 release (125%) and ETBR protein (50%), p < 0.05. Ang1–7 increased NO production (257%) in a Mas and ETBR-dependent manner. Mas and ETBR interaction was observed by immunoprecipitation. To characterise physical interaction between Mas/ETBR, we utilised novel technology, employing peptides scanning the MasR sequence, to define sites of ETBR binding. Mutagenesis identified regions on MasR that confer specificity for ETBR. Peptide disruptors were used for in vitro validation. We previously demonstrated in HMEC that Ang1–7 stimulates Akt phosphorylation (180%), an effect inhibited by peptide disruptors, p < 0.05. To investigate pathophysiological significance, we investigated whether Ang1–7 treatment ameliorates PH. Hypoxia was used to induce PH in mice: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with Ang1–7 30 µg/kg/day. In HP mice, RVSP (18.7 NC vs. 47.6 mmHg HP, p < 0.05) RVH (0.19 NC vs. 0.28 HP, p < 0.01) and ET-1 levels (0.8 NC vs 2.4 pg/ml HP, p < 0.05) were increased and blocked by Ang1–7. Hypercontractility and endothelial dysfunction in HP mice was attenuated by Ang1–7. These findings indicate that vasoprotective effects of Ang1–7 may be mediated through MAS: ETBR dimerization. In conclusion we have identified a novel link between Ang1–7 and ET-1 through physical interactions between MAS and ETBR.