Novel RAB27A Variant Associated with Late-Onset Hemophagocytic Lymphohistiocytosis Alters Effector Protein Binding.

Autosomal recessive mutations in RAB27A are associated with Griscelli syndrome type 2 (GS2), characterized by hypopigmentation and development of early-onset, potentially fatal hemophagocytic lymphohistiocytosis (HLH). We describe a 35-year old male who presented with recurrent fever, was diagnosed with Epstein-Barr virus-driven chronic lymphoproliferation, fulfilled clinical HLH criteria, and who carried a novel homozygous RAB27A c.551G > A p.(R184Q) variant. We aimed to evaluate the contribution of the identified RAB27A variant in regard to the clinical phenotype as well as cellular and biochemical function. The patient displayed normal pigmentation as well as RAB27A expression in blood-derived cells. However, patient NK and CD8+ T cell exocytosis was low. Ectopic expression of the RAB27A p.R184Q variant rescued melanosome distribution in mouse Rab27a-deficient melanocytes, but failed to increase exocytosis upon reconstitution of human RAB27A-deficient CD8+ T cells. Mechanistically, the RAB27A p.R184Q variant displayed reduced binding to SLP2A but augmented binding to MUNC13-4, two key effector proteins in immune cells. MUNC13-4 binding was particularly strong to an inactive RAB27A p.T23N/p.R184Q double mutant. RAB27A p.R184Q was expressed and could facilitate melanosome trafficking, but did not support lymphocyte exocytosis. The HLH-associated RAB27A variant increased Munc13-4 binding, potentially representing a novel mode of impairing RAB27A function selectively in hematopoietic cells.

A novel membrane targeting domain mediates the endosomal or Golgi localization specificity of small GTPases Rab22 and Rab31.

Rab22 and Rab31 belong to the Rab5 subfamily of GTPases that regulates endocytic traffic and endosomal sorting. Rab22 and Rab31 (a.k.a. Rab22b) are closely related and share 87% amino acid sequence similarity, but they show distinct intracellular localization and function in the cell. Rab22 is localized to early endosomes and regulates early endosomal recycling, while Rab31 is mostly localized to the Golgi complex with only a small fraction in the endosomes at steady state. The specific determinants that affect this differential localization, however, are unclear. In this study, we identify a novel membrane targeting domain (MTD) consisting of the C-terminal hypervariable domain (HVD), interswitch loop (ISL), and N-terminal domain as a major determinant of endosomal localization for Rab22 and Rab31, as well as Rab5. Rab22 and Rab31 share the same N-terminal domain, but we find Rab22 chimeras with Rab31 HVD exhibit phenotypic Rab31 localization to the Golgi complex, while Rab31 chimeras with the Rab22 HVD localize to early endosomes, similar to wildtype Rab22. We also find that the Rab22 HVD favors interaction with the early endosomal effector protein Rabenosyn-5, which may stabilize the Rab localization to the endosomes. The importance of effector interaction in endosomal localization is further demonstrated by the disruption of Rab22 endosomal localization in Rabenosyn-5 knockout cells and by the shift of Rab31 to the endosomes in Rabenosyn-5-overexpressing cells. Taken together, we have identified a novel MTD that mediates localization of Rab5 subfamily members to early endosomes via interaction with an effector such as Rabenosyn-5.

Probing the Peculiarity of EhRabX10, a pseudoRab GTPase, from the Enteric Parasite Entamoeba histolytica through In Silico Modeling and Docking Studies.

Entamoeba histolytica (Eh) is a pathogenic eukaryote that often resides silently in humans under asymptomatic stages. Upon indeterminate stimulus, it develops into fulminant amoebiasis that causes severe hepatic abscesses with 50% mortality. This neglected tropical pathogen relies massively on membrane modulation to flourish and cause disease; these modulations range from the phagocytic mode for food acquisition to a complex trogocytosis mechanism for tissue invasion. Rab GTPases form the largest branch of the Ras-like small GTPases, with a diverse set of roles across the eukaryotic kingdom. Rab GTPases are vital for the orchestration of membrane transport and the secretory pathway responsible for transporting the pathogenic effectors, such as cysteine proteases (EhCPs) which help in tissue invasion. Rab GTPases thus play a crucial role in executing the cytolytic effect of E. histolytica. First, they interact with Gal/Nac lectins required for adhering to the host cells, and then, they assist in the secretion of EhCPs. Additionally, amoebic Rab GTPases are vital for encystation because substantial vesicular trafficking is required to create dormant amoebic cysts. These cysts are the infective agent and help to spread the disease. The absence of a "bonafide" vesicular transport machinery in Eh and the existence of a diverse repertoire of amoebic Rab GTPases (EhRab) hint at their contribution in supporting this atypical machinery. Here, we provide insights into a pseudoRab GTPase, EhRabX10, by performing physicochemical analysis, predictive 3D structure modeling, protein-protein interaction studies, and in silico molecular docking. Our group is the first one to classify EhRabX10 as a pseudoRab GTPase with four nonconserved G-motifs. It possesses the basic fold of the P-loop containing nucleotide hydrolases. Through this in silico study, we provide an introduction to the characterization of the atypical EhRabX10 and set the stage for future explorations into the mechanisms of nucleotide recognition, binding, and hydrolysis employed by the pseudoEhRab GTPase family.

Structural and functional study of Legionella pneumophila effector RavA.

Legionella pneumophila is an intracellular pathogen that causes Legionnaire's disease in humans. This bacterium can be found in freshwater environments as a free-living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella-containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER-derived secretory vesicles and membranes forming the Legionella-containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α-helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C-terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C-terminal sequence C409 WTSFCGLF417 . Yeast-two-hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.

Structural dynamics and allostery of Rab proteins: strategies for drug discovery and design.

Rab proteins represent the largest family of the Rab superfamily guanosine triphosphatase (GTPase). Aberrant human Rab proteins are associated with multiple diseases, including cancers and neurological disorders. Rab subfamily members display subtle conformational variations that render specificity in their physiological functions and can be targeted for subfamily-specific drug design. However, drug discovery efforts have not focused much on targeting Rab allosteric non-nucleotide binding sites which are subjected to less evolutionary pressures to be conserved, hence are likely to offer subfamily specificity and may be less prone to undesirable off-target interactions and side effects. To discover druggable allosteric binding sites, Rab structural dynamics need to be first incorporated using multiple experimentally and computationally obtained structures. The high-dimensional structural data may necessitate feature extraction methods to identify manageable representative structures for subsequent analyses. We have detailed state-of-the-art computational methods to (i) identify binding sites using data on sequence, shape, energy, etc., (ii) determine the allosteric nature of these binding sites based on structural ensembles, residue networks and correlated motions and (iii) identify small molecule binders through structure- and ligand-based virtual screening. To benefit future studies for targeting Rab allosteric sites, we herein detail a refined workflow comprising multiple available computational methods, which have been successfully used alone or in combinations. This workflow is also applicable for drug discovery efforts targeting other medically important proteins. Depending on the structural dynamics of proteins of interest, researchers can select suitable strategies for allosteric drug discovery and design, from the resources of computational methods and tools enlisted in the workflow.

The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members.

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.

Antiparasitic dibenzalacetone inhibits the GTPase activity of Rab6 protein of Leishmania donovani (LdRab6), a potential target for its antileishmanial effect.

Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.

Charcot-Marie-Tooth Type 2B: A New Phenotype Associated with a Novel RAB7A Mutation and Inhibited EGFR Degradation.

The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking difficulties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A>G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family.

Genetic and structural studies of RABL3 reveal an essential role in lymphoid development and function.

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.

Rab7 controls innate immunity by regulating phagocytosis and antimicrobial peptide expression in Chinese mitten crab.

The Rab family is the most significant subfamily of small GTP-binding proteins. These proteins have widespread intracellular localization and play an important role in many biological processes. Rab7 plays a crucial role in the innate immune system of crustaceans. In the present study, we cloned and characterized Rab7 from Chinese mitten crab (Eriocheir sinensis), designated EsRab7. The full-length of the EsRab7 cDNA sequence is 1,257 bp and contains a 618-bp open reading frame encoding a 205-amino acid polypeptide. Bioinformatics analysis showed that the Rab7 protein was highly conserved during evolution. Quantitative real-time PCR showed the highest tissue expression in muscle, followed by hepatopancreas. EsRab7 was significantly upregulated in hemocytes after stimulation by Gram-positive Staphylococcus aureus or Gram-negative Vibrio parahaemolyticus. Further studies showed that EsRab7 knockdown during bacterial stimulation resulted in decreased bacterial phagocytosis. In addition, EsRab7 regulated the expression of antimicrobial peptides via the Toll signaling pathway. Collectively, these results demonstrate that EsRab7 plays critical roles in antimicrobial function in the Chinese mitten crab.

TBC1D24-TLDc-related epilepsy exercise-induced dystonia: rescue by antioxidants in a disease model.

Genetic mutations in TBC1D24 have been associated with multiple phenotypes, with epilepsy being the main clinical manifestation. The TBC1D24 protein consists of the unique association of a Tre2/Bub2/Cdc16 (TBC) domain and a TBC/lysin motif domain/catalytic (TLDc) domain. More than 50 missense and loss-of-function mutations have been described and are spread over the entire protein. Through whole genome/exome sequencing we identified compound heterozygous mutations, R360H and G501R, within the TLDc domain, in an index family with a Rolandic epilepsy exercise-induced dystonia phenotype (http://omim.org/entry/608105). A 20-year long clinical follow-up revealed that epilepsy was self-limited in all three affected patients, but exercise-induced dystonia persisted into adulthood in two. Furthermore, we identified three additional sporadic paediatric patients with a remarkably similar phenotype, two of whom had compound heterozygous mutations consisting of an in-frame deletion I81_K84 and an A500V mutation, and the third carried T182M and G511R missense mutations, overall revealing that all six patients harbour a missense mutation in the subdomain of TLDc between residues 500 and 511. We solved the crystal structure of the conserved Drosophila TLDc domain. This allowed us to predict destabilizing effects of the G501R and G511R mutations and, to a lesser degree, of R360H and potentially A500V. Next, we characterized the functional consequences of a strong and a weak TLDc mutation (TBC1D24G501R and TBC1D24R360H) using Drosophila, where TBC1D24/Skywalker regulates synaptic vesicle trafficking. In a Drosophila model neuronally expressing human TBC1D24, we demonstrated that the TBC1D24G501R TLDc mutation causes activity-induced locomotion and synaptic vesicle trafficking defects, while TBC1D24R360H is benign. The neuronal phenotypes of the TBC1D24G501R mutation are consistent with exacerbated oxidative stress sensitivity, which is rescued by treating TBC1D24G501R mutant animals with antioxidants N-acetylcysteine amide or α-tocopherol as indicated by restored synaptic vesicle trafficking levels and sustained behavioural activity. Our data thus show that mutations in the TLDc domain of TBC1D24 cause Rolandic-type focal motor epilepsy and exercise-induced dystonia. The humanized TBC1D24G501R fly model exhibits sustained activity and vesicle transport defects. We propose that the TBC1D24/Sky TLDc domain is a reactive oxygen species sensor mediating synaptic vesicle trafficking rates that, when dysfunctional, causes a movement disorder in patients and flies. The TLDc and TBC domain mutations' response to antioxidant treatment we observed in the animal model suggests a potential for combining antioxidant-based therapeutic approaches to TBC1D24-associated disorders with previously described lipid-altering strategies for TBC domain mutations.

Using two-dimensional convolutional neural networks for identifying GTP binding sites in Rab proteins.

Deep learning has been increasingly and widely used to solve numerous problems in various fields with state-of-the-art performance. It can also be applied in bioinformatics to reduce the requirement for feature extraction and reach high performance. This study attempts to use deep learning to predict GTP binding sites in Rab proteins, which is one of the most vital molecular functions in life science. A functional loss of GTP binding sites in Rab proteins has been implicated in a variety of human diseases (choroideremia, intellectual disability, cancer, Parkinson's disease). Therefore, creating a precise model to identify their functions is a crucial problem for understanding these diseases and designing the drug targets. Our deep learning model with two-dimensional convolutional neural network and position-specific scoring matrix profiles could identify GTP binding residues with achieved sensitivity of 92.3%, specificity of 99.8%, accuracy of 99.5%, and MCC of 0.92 for independent dataset. Compared with other published works, this approach achieved a significant improvement. Throughout the proposed study, we provide an effective model for predicting GTP binding sites in Rab proteins and a basis for further research that can apply deep learning in bioinformatics, especially in nucleotide binding site prediction.

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering.

Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g. Rab/lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction-monitoring (MRM) analysis, to assess the differential expression of small GTPases in MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method displayed superior sensitivity and reproducibility over the shotgun-proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with >1.5-fold change), respectively, in the tamoxifen-resistant line relative to in the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role for RAB31 in tamoxifen resistance in ER-positive breast-cancer cells. Together, our results demonstrated that the targeted proteomic method constituted a powerful approach for revealing the role of small GTPases in therapeutic resistance.

Integrative functional genomics identifies regulatory genetic variant modulating RAB31 expression and altering susceptibility to breast cancer.

Despite the successes of genome-wide association study (GWAS) in identifying breast cancer (BC) risk-associated variants, only a small fraction of the heritability can be explained. The greatest challenge in the post-GWAS is to identify causal variants and underlying mechanisms responsible for BC susceptibility. In this study, we integrated functional genomic data from ENCODE ChIP-seq, ANNOVAR, and the TRANSFAC matrix to identify potentially regulatory variants with modulating FOXA1-binding affinity across the whole genome, and then conducted a two-stage case-control study including 2164 cases and 2382 controls to investigate the associations between candidate SNPs and BC susceptibility. We identified a BC susceptibility SNP, rs6506689 G>T, with an odds ratio (OR) of 1.23 (95% confidence interval = 1.07-1.40, P = 0.003) under a dominant model in the combined study. Biological assays indicated that the germline G>T variation at rs6506689 creates a FOXA1-binding site and up-regulates the expression of RAB31, thus playing an important role in the development of BC. Our results highlight the importance of regulatory genetic variants in the development of BC by influencing TF-DNA interaction and provide critical insights to pinpoint causal genetic variants.

Allosteric binding sites in Rab11 for potential drug candidates.

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously.

A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy.

Rab GTPases are key regulators of membrane trafficking, and many are activated by guanine nucleotide exchange factors bearing a differentially expressed in normal and neoplastic cells (DENN) domain. By activating the small GTPase Rab12, DENN domain-containing protein 3 (DENND3) functions in autophagy. Here, we identified a structural domain (which we name PHenn) containing a pleckstrin homology subdomain that binds actin and is required for DENND3 function in autophagy. We found that a hydrophobic patch on an extended ß-turn of the PHenn domain mediates an intramolecular interaction with the DENN domain of DENND3. We also show that DENND3 binds actin through a surface of positively charged residues on the PHenn domain. Substitutions that blocked either DENN or actin binding compromised the role of DENND3 in autophagy. These results provide new mechanistic insight into the structural determinants regulating DENND3 in autophagy and lay the foundation for future investigations of the DENN protein family.

Roles for RAB24 in autophagy and disease.

Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.

Molecular control of Rab activity by GEFs, GAPs and GDI.

Rab proteins are the major regulators of vesicular trafficking in eukaryotic cells. Their activity can be tightly controlled within cells: Regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), they switch between an active GTP-bound state and an inactive GDP-bound state, interacting with downstream effector proteins only in the active state. Additionally, they can bind to membranes via C-terminal prenylated cysteine residues and they can be solubilized and shuttled between membranes by chaperone-like molecules called GDP dissociation inhibitors (GDIs). In this review we give an overview of Rab proteins with a focus on the current understanding of their regulation by GEFs, GAPs and GDI.