Biology subjects

Cota, E.

Publications and source records attributed to Cota, E..

4 recordsLinked to original sources

bioRxiv2026

A potent macrocyclic peptide binder enables effector-free crystallization of the GTPase RAB27A

RAB27A is a member of a highly conserved family of small GTPases that regulate intracellular vesicle trafficking. Multiple studies have linked RAB27A cellular activity to exosome-mediated modulation of the tumour microenvironment and inflammatory signalling, highlighting it as an attractive target for drug discovery. However, identifying structurally validated and selective inhibitors of the RAB27A-effector interaction has proven challenging, partly because wild-type RAB27A crystallizes only in complex with effector proteins such as Slp2a. Using mRNA display, we identified macrocyclic peptide ligands that target the RAB27A effector-binding interface. Optimisation of initial hits led to IMP2660, a nanomolar-affinity RAB27A binder that effectively disrupts RAB27A-effector interactions. IMP2660 enabled determination of a 2.1[A] structure of active-state RAB27A bound to a noncovalent macrocyclic peptide in the absence of an effector, revealing structural features of the effector-binding surface and providing a platform for future structure-guided ligand development.

biochemistry↗

bioRxiv2026

Discovery of stereoselective targeted covalent inhibitors of the RAB27-effector protein-protein interaction

RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B play roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site, and large RAB27-effector protein-protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B-Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be toxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 which can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.

biochemistry↗

bioRxiv2023

A RabGAP-Rab GTPase pair regulates plant autophagy and immunity

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 an important 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 from Nicotiana benthamiana, but not its mutated form deficient in ATG8 binding, reduced autophagic flux in N. benthamiana and Arabidopsis. Furthermore, Rab3GAPL-knockout mutants of the liverwort Marchantia polymorpha exhibited enhanced autophagic flux under both normal and heat stress conditions, suggesting that Rab3GAPLs 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.

plant biology↗

bioRxiv2018

A fungal ribonuclease-like effector protein inhibits plant host ribosomal RNA degradation

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. Proteins with a predicted ribonuclease (RNase)-like fold (termed RALPHs) comprise the largest set of secreted effector candidates within the B. graminis f. sp. hordei genome. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in monocotyledenous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with five host proteins: two translation elongation factors (eEF1 and eEF1{gamma}), two pathogenesis-related proteins (PR5 and PR10) and a glutathione-S-transferase. We present the first crystal structure of a RALPH, CSEP0064/BEC1054, demonstrating it has an RNase-like fold. The protein interacts with total RNA and weakly with DNA. Methyl jasmonate levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose that CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins that would otherwise lead to host cell death, an unviable interaction and demise of the fungus.

plant biology↗