Thomas R. Hinds

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

Hippocampal Neurotoxicity of Δ9-Tetrahydrocannabinol

Marijuana consumption elicits diverse physiological and psychological effects in humans, including memory loss. Here we report that Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is toxic for hippocampal neurons. Treatment of cultured neurons or hippocampal slices with THC caused shrinkage of neuronal cell bodies and nuclei as well as genomic DNA strand breaks, hallmarks of neuronal apoptosis. Neuron death induced by THC was inhibited by nonsteroidal anti-inflammatory drugs, including indomethacin and aspirin, as well as vitamin E and other antioxidants. Furthermore, treatment of neurons with THC stimulated a significant increase in the release of arachidonic acid. We hypothesize that THC neurotoxicity is attributable to activation of the prostanoid synthesis pathway and generation of free radicals by cyclooxygenase. These data suggest that some of the memory deficits caused by cannabinoids may be caused by THC neurotoxicity.

Cyclic nucleotide analogs as probes of signaling pathways

To the editor: Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers that regulate multiple targets including different cAMPor cGMP-dependent protein kinases (PKAs, PKGs)1,2, exchange proteins directly activated by cAMP (Epacs)3, phosphodiesterases (PDEs)4 and cyclic nucleotide-gated ion channels. Cyclic nucleotide analogs are widely used to study specificity of cellular signaling mediated by these target proteins. However, the selectivities and stabilities of these analogs need to be fully understood to properly interpret results and rigorously assess the mechanisms by which these analogs work in the cell. To better understand the selectivity and cross-reactivity of these analogs, we measured the activation or inhibitory activity of 13 commonly used cyclic nucleotide analogs with isozymes of PKA, PKG and Epac (Table 1), and with 8 different PDEs (Table 2 and Supplementary Tables 1 and 2 online). To measure their stability against hydrolysis, we used isothermal microcalorimetry5, a method that allowed us to evaluate whether or not an analog can function as a substrate or inhibitor for PDEs. We found that indeed some of these analogs were hydrolyzed by multiple PDEs, and other analogs were competitive inhibitors of PDEs. Here we provide half-maximal inhibition constant (Ki) data for all of the non-hydrolyzable analogs, and MichaelisMenten constant (Km) and maximum velocity (Vmax) values for all of the hydrolyzable analogs. Each of these values as well as the analog’s mode of inhibition can be determined in a single experiment (Table 2, Supplementary Methods and Supplementary Figures 1–5 online). The data strongly implied that several of these analogs might, in addition to their primary effects, also cause elevation of cAMP or cGMP indirectly by inhibiting PDEs in the cell. Such an effect could cloud interpretation of the use of these analogs. Similarly, analogs that are PDE substrates also might have their duration of action substantially reduced. To illustrate this point we showed that Sp-8-pCPT-2′O-Me-cAMPS, a highly specific, non-hydrolyzable Epac activator in vitro, can under certain conditions enhance cGMP-PKG and cAMPPKA signaling pathways in intact platelets (Supplementary Fig. 1). Specifically, we observed enhanced phosphorylation of vasodialatorstimulated phosphoprotein (VASP) at both PKA and PKG phosphorylation sites after the addition of Sp-8-pCPT-2′-O-Me-cAMPS. These data indicate that this ‘selective Epac activator’ is able to indirectly activate the cAMP-PKA and cGMP-PKG signaling pathways presumably through inhibition of platelet PDE5 and/or PDE3 (Supplementary Methods and Supplementary Discussion online). We also list in vitro selectivity data for all of the presently available commonly used cyclic nucleotide analogs for different forms of PKA, PKG and Epac I (Table 1). Data for several of these analogs have not

Ion transport ATPases as targets for free radical damage: Protection by an aminosteroid of the Ca2+ pump atpase and Na+/K+ pump ATPase of human red blood cell membranes

Preincubation of red blood cell membranes in the presence of ferrous sulfate and EDTA resulted in both a concentration- and time-dependent inhibition of the Na+/K+ pump ATPase, basal Ca2+ pump ATPase, and the calmodulin- (CaM) activated Ca2+ pump ATPase. The IC50 for all three ATPases was approximately 2.5 x 10(-5) M iron. The addition to membranes of ferrous iron and EDTA in an approximately 1:1 ratio resulted in conversion to the ferric iron form in several minutes. However, inhibition of the ion pump ATPases and cross-linking of membrane proteins occurred over the course of several hours. The time course of formation of thiobarbituric acid-reactive substances (TBARS) closely paralleled inhibition of the ion pump ATPases. Inhibition of the ion pump ATPases was prevented by the addition of deferoxamine or superoxide dismutase but not by mannitol, or catalase. Both butylated hydroxytoluene and tirilazad mesylate (U74006F) prevented the formation of TBARS, limited the inhibition of the ion pump ATPases, and reduced cross-linking of membrane proteins. These data may be interpreted to suggest that inhibition of ion pump ATPases in plasma membranes may occur as a result of iron-promoted formation of superoxide and subsequent lipid peroxidation, which can be prevented by free-radical scavengers including butylated hydroxytoluene and U74006F.

Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101; however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination

Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.

SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFBXL3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3–SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein–substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

Plasma membrane Ca2+ transport: Stimulation by soluble proteins

Abstract Inside-out membrane vesicles were prepared from human red blood cells. In the presence of ATP, these vesicles took up 45 Ca 2+ against a chemical gradient. The active transport of Ca 2+ was increased by addition of an activator protein of (Ca 2+ +Mg 2+ )-ATPase isolated from the membrane-free hemolysate of human red blood cells. A closely related protein, the protein modulator of cyclic AMP phosphodiesterase from bovine brain, also increased the rate of active transport of 45 Ca 2+ . Addition of the calcium ionophore A23187 caused a rapid efflux of 45 Ca 2+ from loaded, inside-out vesicles. When La 3+ was added to the system in the presence of activator protein, the uptake of 45 Ca 2+ was inhibited. Results are compatible with the interpretation that activity of the plasma membrane Ca 2+ pump may be modulated by certain cytoplasmic proteins.

Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals: Protection by 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7h-pyrrolo[2,3-d]pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger

Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 microM ferrous sulfate and 200 microM EDTA, Fe2+/EDTA) resulted inhibition of the Na+/K+ -pump ATPases was also associated with membrane protein crosslinking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 microM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U-89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U-89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes.

CALMODULIN AND THE PLASMA MEMBRANE CALCIUM PIIMP

The data summarized and presented in this paper are consistent with the interpretation that CaM participates in the regulation of the plasma membrane calcium pump. Certain drugs, such as phenothiazines can antagonize CaM. Ca2+ loading of RBCs promotes CaM binding to RBC membranes and results in decreased responsiveness of the [Ca2+ + Mg2+)-ATPase to CaM. The latter effect may be mediated by a Ca2+ activated transglutaminase. Activation of (Ca2+ + Mg2+)-ATPase by CaM in vitro was shown not to be instantaneous, probably because of slow binding. CaM binding to isolated RBC membranes exhibits a Ca2+ dependence that is similar to that for activation of the (Ca2+ + Mg2+)-ATPase, and CaM binding does not decrease at high [Ca2+]s. Calculations based on assumed values for RBC [Ca2+], [CaM], and binding affinities of Ca2+ for CaM and CaM(Ca2+)n for the Ca2+ pump ATPase resulted in the tentative conclusion that most pump sites are occupied by CaM in the RBC in vivo. This conclusion, and the relatively slow time course of the CaM effect on (Ca2+ + Mg2+)-ATPase prompt us to suggest that, for all practical purposes, CaM is a subunit of the Ca2+ pump ATPase in vivo.