Kokoro Hayashi

Regulation of Rice NADPH Oxidase by Binding of Rac GTPase to Its N-Terminal Extension

Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91phox and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern–induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac–Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac–Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac–Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.

14-3-3 proteins act as intracellular receptors for rice Hd3a florigen

‘Florigen’ was proposed 75 years ago to be synthesized in the leaf and transported to the shoot apex, where it induces flowering. Only recently have genetic and biochemical studies established that florigen is encoded by FLOWERING LOCUS T (FT), a gene that is universally conserved in higher plants. Nonetheless, the exact function of florigen during floral induction remains poorly understood and receptors for florigen have not been identified. Here we show that the rice FT homologue Hd3a interacts with 14-3-3 proteins in the apical cells of shoots, yielding a complex that translocates to the nucleus and binds to the Oryza sativa (Os)FD1 transcription factor, a rice homologue of Arabidopsis thaliana FD. The resultant ternary ‘florigen activation complex’ (FAC) induces transcription of OsMADS15, a homologue of A. thaliana APETALA1 (AP1), which leads to flowering. We have determined the 2.4 Å crystal structure of rice FAC, which provides a mechanistic basis for florigen function in flowering. Our results indicate that 14-3-3 proteins act as intracellular receptors for florigen in shoot apical cells, and offer new approaches to manipulate flowering in various crops and trees.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca2+ binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca2+ binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

pCold-GST vector: A novel cold-shock vector containing GST tag for soluble protein production

The production of recombinant protein in Escherichia coli is often hampered by low expression levels and low solubility. A variety of methodologies have been developed including protein production at low temperature, and fusion protein expression using soluble protein tags. Here, we present the novel cold-shock vector pCold-GST for high-level expression of soluble proteins in E. coli. This vector is a modified pCold I cold-shock vector that includes the glutathione S-transferase (GST) tag. The pCold-GST expression system developed was applied to 10 proteins that could not be expressed using conventional E. coli expression methodologies, and nine of these proteins were successfully obtained in the soluble fraction. The expression and purification of two unstable protein fragments were also demonstrated by employing a C-terminal hexa-histidine tag for purification purposes. The purified proteins were amenable to NMR analyses. These data suggest that the pCold-GST expression system can be utilized to improve the expression and purification of various proteins.

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1H-15N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

Crystallographic characterization of the N-terminal domain of a plant NADPH oxidase

Respiratory burst oxidase homologue (Rboh), which is found in the plasma membrane, is a generator of reactive oxygen species (ROS) in plants. Many studies have indicated that the ROS produced by Rboh play critical roles in various cellular activities, including plant defence against pathogens. Crystals of the N-terminal domain of Oryza sativa RbohB (OsRbohB) have been obtained. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.4, b = 72.2, c = 118.9 A. An intensity data set was collected to 2.4 A resolution.

Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor

Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function.

Structural basis for floral induction by rice florigen Hd3a

A Sulfolobus solfataricus acylpeptide hydrolase (APEH), named APEHSs has been identified and characterized. The protein is the endogenous protease target of the already isolated inhibitor SsCEI [1]. APEH is one of the four members of the prolyl oligopeptidase family, which removes acylated amino acid residues from the N terminus of blocked oligopeptides. SsCEI is the first protein able to efficiently inhibit APEH from mammalian sources with IC50 values in the nanomolar range. The 3D model of APEHSs alone or in complex with SsCEI has been proposed, suggesting an inhibition mechanism of steric blockage on substrate access to the active site or on product release [2]. Interestingly, the identified reactive site loop (RSL) of SsCEI includes an unusual amino acid sequence which cannot be classified in any of the canonical motifs of serine protease inhibitors so far characterized. Therefore, a number of small peptides has been designed and synthesized on the basis of the RSL sequence. They are surprisingly stable and highly structured in aqueous solutions and show inhibitory activity against S. solfataricus, and human APEHs with IC50 values in the order of low micromolar. In light of a recently proposed cooperative role for the APEH–proteasome enzymatic system in controlling protein turnover, we investigated the proteasome down-regulation via APEH inhibition, hypothesizing that APEH can be used as a new target to indirectly control/modulate proteasome functions in tumoral cells. We identified different compounds that induce proteasome down-regulation via APEH, in both cell-free and cell-based assays. These molecules efficiently down-regulated human APEH activity in a dose-dependent manner in the human colon carcinoma cell line (Caco-2 cells) or in the baby-hamster-kidney (BHK) cell line, without any toxic effects and inducing proapoptotic and antitumoral effects. Surprisingly, in both cell lines such molecules markedly reduced the proteasome activity with a concomitant accumulation of several known cytoplasmic proteasome substrates. Furthermore, a molecular docking analysis has been carried out to assess the potential enzyme binding sites involved in the APEH–inhibitors interactions. These results represent a starting point for a promising strategy in cancer therapy involving a new class of molecules for proteasome down-regulation mediated by knock-out of APEH activity.