A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

Identification of the first structurally validated covalent ligands of the small GTPase RAB27A.

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket ('WF-binding pocket') via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a.

Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Šresolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms.

Structural and functional characterization of a frataxin from a thermophilic organism.

Frataxins form an interesting family of iron-binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron-sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X-ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation in the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable among the frataxin orthologues. We also characterized the iron-binding mode of Ct frataxin and demonstrated that it binds it through a semiconserved negatively charged ridge on the first helix and beta-strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron-sulfur cluster synthesis, and act as its inhibitor.

Direct Targeting of the Ras GTPase Superfamily Through Structure- Based Design.

The Ras superfamily of small monomeric GTPases includes some of the most prominent cancer targets for which no selective therapeutic agent has yet been successfully developed. The turn of the millennium saw a resurgence of efforts to target these enzymes using new and improved biophysical techniques to overcome the perceived difficulties of insurmountably high affinity for guanosine nucleotides and flat, flexible topology lacking suitable pockets for small molecule inhibitors. Further, recent investigations have begun to probe the dynamic conformational status of GTP-bound Ras, opening up new mechanisms of inhibition. While much of the literature has focused on the oncogenic Ras proteins, particularly K-Ras, these represent only a small minority of therapeutically interesting targets within the superfamily; for example, the Rab GTPases are the largest subfamily of about 70 members, and present an as yet untapped class of potential targets. The present review documents the key methodologies employed to date in structure-guided attempts to drug the Ras GTPases, and forecasts their transferability to other similarly challenging proteins in the superfamily.

Comparing the calcium binding abilities of two soybean calmodulins: towards understanding the divergent nature of plant calmodulins.

The discovery that plants contain multiple calmodulin (CaM) isoforms of variable sequence identity to animal CaM suggested an additional level of sophistication in the intracellular role of calcium regulation in plants. Past research has focused on the ability of conserved or divergent plant CaM isoforms to activate both mammalian and plant protein targets. At present, however, not much is known about how these isoforms respond to the signal of an increased cytosolic calcium concentration. Here, using isothermal titration calorimetry and NMR spectroscopy, we investigated the calcium binding properties of a conserved (CaM1) and a divergent (CaM4) CaM isoform from soybean (Glycine max). Both isoforms bind calcium with a semisequential pathway that favors the calcium binding EF-hands of the C-terminal lobe over those of the N-terminal lobe. From the measured dissociation constants, CaM4 binds calcium with a threefold greater affinity than CaM1 (K(d,Ca,mean) of 5.0 versus 14.9 µM) but has a significantly reduced selectivity against the chemically similar magnesium cation that binds preferentially to EF-hand I of both isoforms. The implications of a potential magnesium/calcium competition on the activation of CaM1 and CaM4 are discussed in context with their ability to respond to stimulus-specific calcium signatures and their known physiological roles.

Structural analysis of a calmodulin variant from rice: the C-terminal extension of OsCaM61 regulates its calcium binding and enzyme activation properties.

OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca(2+)] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca(2+). NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca(2+)-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca(2+) binding affinity of EF-hands III and IV in OsCaM61. In Ca(2+)-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser(172) and Ala(173), residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca(2+) signaling events.

Correlation between inducible nitric oxide synthase and cyclooxygenase-2 expression in human colorectal adenocarcinoma: a cross-sectional study.

Cyclooxygenase-2 (COX-2) enzyme is believed to play a role in tumor angiogenesis, differentiation, and apoptosis. The inducible isoform of nitric oxide synthase (iNOS) also has the potential ability to damage DNA and conceivably contribute to tumor formation by a rise in nitric oxide production. Seventeen patients diagnosed with colorectal adenocarcinoma, who underwent surgical resection of the tumor, were enrolled in the study. Two macroscopic tissue samples, one from the tumor and the other from the tumor free surgical margin were collected from every patient as formalin fixed paraffin embedded blocks. Samples were analyzed for iNOS and COX-2 expression by immunohistochemistry and Western blotting. Results were digitized and semi-quantitatively analyzed. Immunohistochemistry revealed a similar pattern of expression for both iNOS and COX-2, as both were detected in tumor and epithelial cells. The mean iNOS and COX-2 levels determined by Western blotting method were significantly higher in tumor than in the tumor-free tissues (Wilcoxon signed-rank test, p < 0.001 both for iNOS and COX-2). Patients with lymph node involvement had higher levels of both enzymes in tumors (Mann-Whitney U test, p < 0.05). There was correlation between iNOS and COX-2 expression of tumor determined by immunohistochemistry and also by Western blotting (Spearman's rho test, R = 0.53, p = 0.03 and R = 0.57, p = 0.02, respectively). In conclusion, our results point out a relationship between iNOS and COX-2 expression in human colorectal adenocarcinomas and may also suggest a possible link between advanced stages of the disease and higher expression of iNOS and COX-2.