MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.     

A Mechanism of Global Shape-dependent Recognition and Phosphorylation of Filamin by Protein Kinase A

Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by ∼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser²¹⁵² site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser²¹⁵². These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.     

Permian and Triassic Radiolaria from Northwest Thailand: paleogeographical implications

Well-preserved radiolarians were recovered from seven sections in the Mae Hong Son-Mae Sariang area, northwestern Thailand. 51 species assigned to 34 genera are identified, including 1 new species (Triassospongosphaera erici Feng sp. nov.) and 19 unidentified species. They are divided into the Late Permian, late Ladinian and middle Carnian radiolarian assemblages. Newly identified radiolarian assemblages, together with the published radiolarian biostratigraphic data from this region, indicate that there was a pelagic basin during the Late Paleozoic and Triassic. This basin was joined to the Chiang Dao and Changning-Menglian oceanic basins, and they represent the main oceanic basin of the Paleotethyan Archipelago Ocean. This main oceanic basin was situated in the traditional “Shan-Thai Block”. Therefore, “the Shan-Thai Block” was not a single block during that stage, but composed of the Paleotethyan Ocean and two continental terranes that were affiliated with the Gondwana and Cathaysian domains, respectively.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor

The Kv-like (potassium voltage-dependent) K⁺ channels at the plasma membrane, including the inward-rectifying KAT1 K⁺ channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K⁺ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K⁺ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.Plant cells utilize the potassium ion (K⁺) to maintain hydrostatic (turgor) pressure, to drive irreversible cell expansion for growth, and to facilitate reversible changes in cell volume during stomatal movements. Potassium uptake and its circulation throughout the plant relies both on high-affinity, H⁺-coupled K⁺ transport (Quintero and Blatt, 1997; Rubio et al., 2008) and on K⁺ channels to facilitate K⁺ ion transfer across cell membranes. Uptake via K⁺ channels is thought to be responsible for roughly 50% of the total K⁺ content of the plant under most field conditions (Spalding et al., 1999; Rubio et al., 2008; Amtmann and Blatt, 2009). K⁺ channels confer on the membranes of virtually every tissue distinct K⁺ conductances and regulatory characteristics (Véry and Sentenac, 2003; Dreyer and Blatt, 2009). Their characteristics are thus of interest for engineering directed to manipulating K⁺ flux in many aspects of plant growth and cellular homeostasis. The control of K⁺ channel gating has been identified as the most promising target for the genetic engineering of stomatal responsiveness (Lawson and Blatt, 2014; Wang et al., 2014a), based on the recent development of quantitative systems models of guard cell transport and metabolism (Chen et al., 2012b; Hills et al., 2012; Wang et al., 2012). By contrast, modifying the expression and, most likely, the population of native K⁺ channels at the membrane was found to have no substantial effect on stomatal physiology (Wang et al., 2014b).The Kv-like K⁺ channels of the plant plasma membrane (Pilot et al., 2003; Dreyer and Blatt, 2009) share a number of structural features with the Kv superfamily of K⁺ channels characterized in animals and Drosophila melanogaster (Papazian et al., 1987; Pongs et al., 1988). The functional channels assemble from four homologous subunits and surround a central transmembrane pore that forms the permeation pathway (Daram et al., 1997). Each subunit comprises six transmembrane α-helices, designated S1 to S6, and both N and C termini are situated on the cytosolic side of the membrane (Uozumi et al., 1998). The pore or P loop between the S5 and S6 α-helices incorporates a short α-helical stretch and the highly conserved amino acid sequence TxGYGD, which forms a selectivity filter for K⁺ (Uozumi et al., 1995; Becker et al., 1996; Nakamura et al., 1997). The carbonyl oxygen atoms of these residues in all four K⁺ channel subunits face inward to form coordination sites for K⁺ ions between them (Doyle et al., 1998; Jiang et al., 2003; Kuo et al., 2003; Long et al., 2005) and a multiple-ion pore (Thiel and Blatt, 1991) such that K⁺ ions pass through the selectivity filter as if in free solution. The plant channels are also sensitive to a class of neurotoxins that exhibit high specificity in binding around the mouth of the channel pore (Obermeyer et al., 1994).These K⁺ channels also share a common gating mechanism. Within each subunit, the first four α-helices form a quasiindependent unit, the voltage sensor domain (VSD), with the S4 α-helix incorporating positively charged (Arg or Lys) residues regularly positioned across the lipid bilayer and transmembrane electric field. Voltage displaces the S4 α-helix within the membrane and couples rotation of the S5 and S6 α-helices lining the pore, thereby opening or closing the channel (Sigworth, 2003; Dreyer and Blatt, 2009). For outward-rectifying channels, such as the mammalian Kv1.2 and the D. melanogaster Shaker K⁺ channels, an inside-positive electric field drives the positively charged, S4 α-helix outward (the up position), which draws on the S4-S5 linker to open the pore. This simple expedient of a lever and string secures current flow in one direction by favoring opening at positive, but not negative, voltages. This same model applies to the Arabidopsis (Arabidopsis thaliana) Kv-like K⁺ channels, including outward rectifiers that exhibit sensitivity to external K⁺ concentration (Blatt, 1988; Blatt and Gradmann, 1997; Johansson et al., 2006), and it serves equally in the gating of inward-rectifying K⁺ channels such as KAT1, which gates open at negative voltages (Dreyer and Blatt, 2009).Studies of KAT1 gating (Latorre et al., 2003; Lai et al., 2005) have indicated that the S4 α-helix of the channel most likely undergoes very similar conformational changes with voltage as those of the mammalian and Shaker K⁺ channels. These findings conform with the present understanding of the evolution of VSD structure (Palovcak et al., 2014) and the view of a common functional dynamic to its molecular design. It is likely, therefore, that a similar electrostatic network occurs in KAT1 to stabilize the VSD. Crucially, however, experimental evidence in support of such a network has yet to surface. Electrostatic countercharges and the hydration of amino acid side chains between the α-helices within the VSDs of mammalian and Shaker K⁺ channel models are important for the latch-like stabilization of the so-called down and up states of these channels (Tao et al., 2010; Pless et al., 2011). Nonetheless, some studies (Gajdanowicz et al., 2009; Riedelsberger et al., 2010) have pointed to subtle differences in the structure of KAT1 that relate to the VSD.We have explored the electrostatic network of the KAT1 VSD through site-directed mutagenesis to manipulate the voltage dependence of KAT1, combining these studies with molecular dynamic simulations previously shown to accommodate the plant VSDs and their hydration during gating transitions (Gajdanowicz et al., 2009; Garcia-Mata et al., 2010). We report here that gating of KAT1 is sensitive to manipulations affecting a set of electrostatic charge transfer centers. These findings conform in large measure to the mammalian and Shaker models. However, virtually all manipulations affecting a highly conserved, central Phe favor the up state of the VSD and the closed KAT1 channel, whereas mutations affecting the electrostatic networks on either side of this Phe favor the down state of the VSD and the open channel. These and additional observations suggest that hydration within the VSD is a major determinant of KAT1 gating.     

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.     

Thyroid ultrasound - a piece of cake?

The introduction of sonographic imaging has revolutionized the diagnostics of thyroid pathologies. Nowadays, thyroid ultrasound examination has become an essential part of routine thyroid gland evaluation. Although one of the greatest advantages of this examination lies in its simplicity, it requires a solid theoretical background, as well as a lot of experience for the examiner to become fluent in adequate interpretation of its results. The aim of this summary is to present a review of the most important aspects of both the technique and interpretation of thyroid ultrasound pictures with regard to the most common difficulties a thyroid sonographer may come across in everyday practice.     

MAGIC Tool: integrated microarray data analysis

Summary: Several programs are now available for analyzing thelarge datasets arising from cDNA microarray experiments. Mostprograms are expensive commercial packages or require expensivethird party software. Some are freely available to academicresearchers, but are limited to one operating system. MicroArrayGenome Imaging and Clustering Tool (MAGIC Tool) is an open sourceprogram that works on all major platforms, and takes users ‘fromtiff to gif’. Several unique features of MAGIC Tool areparticularly useful for research and teaching. Availability: http://www.bio.davidson.edu/MAGIC Contact: laheyer{at}davidson.edu     

Aluminum chloride-mediated kinetic resolution of racemic gamma-substituted-gamma-lactones

Preparation of chiral gamma-substituted-gamma-lactones (1) through kinetic resolution is described. (S)-(-)-1-Phenylethylamine (2) in the presence of anhydrous AlCl(3) shows satisfactory levels of enantioselection in reaction with racemic gamma-substituted-gamma-lactones 1, where (R)-1 remains unreacted, while (S)-1 is enantioselectively converted to the ring-opened amide (S,S)-4. The enantiopurity of (R)-(+)- gamma-substituted gamma-lactones recovered ranges from 62-98% ee.     

Cyclotosaurus cf. PosthumusFraas (Capitosauridae,Stereospondyli) from the Huai Hin Lat Formation (Upper Triassic), Northeastern Thailand: With a note on capitosaurid biogeography

Part of a large capitosaurid skull, similar to that of Cyclotosaurus posthumus from the Upper Triassic of Germany, has been discovered in the upper part of the Huai Hin Lat Formation near Chulabhorn (Nam Phrom) Dam. This discovery is consistent with the presumed Norian age of this formation. Although the phylogeny of the Capitosauridae is still unclear, the group of Upper Triassic Cyclotosaurus species to which C. posthumus belongs is monophyletic and seems to be known only from Laurasia or Northwestern Gondwana (Morocco). The occurrence of C. cf. posthumus in Thailand is consistent with the hypothesis previously put forward, that this part of Southeast Asia was bound to Laurasia in Mesozoic times.