Rab Family Of Small GTPases Research Articles

Structural basis for Rab23 activation and a loss-of-function mutation in Carpenter Syndrome

Rab23 is a member of the Rab family of small GTPases. It plays crucial roles in Hedgehog signaling, ciliary transport, and embryonic development. As a small GTPase, Rab23 cycles between the GDP-bound inactivated state and the GTP-bound activated state. Mutations in Rab23 is directly implicated in Carpenter Syndrome, a development disorder characterized by deformed skulls, abnormal fingers or toes, and intellectual disabilities. Several clinical point mutations, e.g. M12K, C85R and Y79del, have been found to occur within the GTPase domain. However, the mechanisms of activation of Rab23 and pathogenesis of its clinical mutants are still unclear with limited structural information. So far, there are only two reported crystal structures of mouse Rab23 in complex with GDP. Here, we determined high-resolution crystal structures of human Rab23 and the human Rab23 Y79del clinical mutant, in complex with GDP and GMPPNP, a non-hydrolysable GTP analogue, respectively. Supported by in vitro biochemical and functional analyses, we demonstrated that the Y79 deletion mutant exhibited structural distortions in the Switch II region relative to that of the wild-type. The structural changes potentially disrupted the binding of Rab23 Y79del to its interacting partners, thus leading to a loss-of-function and the development of Carpenter Syndrome.

Comprehensive knockout analysis of the RAB family small GTPases reveals an overlapping role of RAB2 and RAB14 in autophagosome maturation

ABSTRACT Macroautophagy, simply referred to below as autophagy, is an intracellular degradation system that is highly conserved in eukaryotes. Since the processes involved in autophagy are accompanied by membrane dynamics, RAB small GTPases, key regulators of membrane trafficking, are generally thought to regulate the membrane dynamics of autophagy. Although more than half of the mammalian RABs have been reported to be involved in canonical and selective autophagy, no consensus has been reached in regard to the role of RABs in mammalian autophagy. Here, we comprehensively analyzed a rab-knockout (KO) library of MDCK cells to reevaluate the requirement for each RAB isoform in basal and starvation-induced autophagy. The results revealed clear alteration of the MAP1LC3/LC3-II level in only four rab-KO cells (rab1-KO, rab2-KO, rab7a-KO, and rab14-KO cells) and identified RAB14 as a new regulator of autophagy, specifically at the autophagosome maturation step. The autophagy-defective phenotype of two of these rab-KO cells, rab2-KO and rab14-KO cells, was very mild, but double KO of rab2 and rab14 caused a severer autophagy-defective phenotype (greater LC3 accumulation than in single-KO cells, indicating an overlapping role of RAB2 and RAB14 during autophagosome maturation. We also found that RAB14 is phylogenetically similar to RAB2 and that it possesses the same properties as RAB2, i.e. autophagosome localization and interaction with the HOPS subunits VPS39 and VPS41. Our findings suggest that RAB2 and RAB14 overlappingly regulate the autophagosome maturation step through recruitment of the HOPS complex to the autophagosome. Abbreviation: AID2: auxin-inducible degron 2; ATG: autophagy related; BafA1: bafilomycin A1; CKO: conditional knockout; EBSS: Earle’s balanced salt solution; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; HRP: horseradish peroxidase; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MDCK: Madin-Darby canine kidney; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; 5-Ph-IAA: 5-phenyl-indole-3-acetic acid; pAb: polyclonal antibody; siRNA: small interfering RNA; SNARE: soluble NSF-attachment protein receptor; TF: transferrin; WT: wild-type.

The Small GTPase Rab7 Regulates Antigen Processing in B Cells in a Possible Interplay with Autophagy Machinery.

In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process.

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

The diagnostic and therapeutic potential of exosomal proteins in breast cancer

Exosomes are membrane vesicles 30–150 nm in size released by cells upon fusion of multivesicular bodies with the plasma membrane. A distinctive feature of these vesicles is the presence of the surface tetraspanins CD9, CD63, and CD81. The Rab family of small GTPases, including Rab27A and Rab27B, controls various steps in exosome release, including transport of multivesicular bodies and fusion of the multivesicular body to the plasma membrane. It is commonly accepted to date that exosomes are the main carriers of information between cells under physiological conditions, such as mammary development and lactation, and under pathological conditions, such as breast cancer. This review considers the peculiarities of exosome formation, secretion and transport, their composition and role in normal and breast cancer, as well as the prospects for using these vesicles to develop early non-invasive diagnostics and improve the effectiveness of anti-tumor therapy.

RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

In-silico drug design for the novel Karachi-NF001 strain of brain-eating amoeba: Naegleria fowleri.

Naegleria fowleri (N. fowleri) is a free-living thermophilic amoeba of fresh water and soil. The amoeba primarily feeds on bacteria but can be transmitted to humans upon contact with freshwater sources. Furthermore, this brain-eating amoeba enters the human body through the nose and travels to the brain to cause primary amebic meningoencephalitis (PAM). N. fowleri has been reported globally since its discovery in 1961. Recently a new strain of N. fowleri named Karachi-NF001 was found in a patient who had traveled from Riyadh, Saudi Arabia to Karachi in 2019. There were 15 unique genes identified in the genome of the Karachi-NF001 strain compared to all the previously reported strains of N. fowleri worldwide. Six of these genes encode well-known proteins. In this study, we performed in-silico analysis on 5 of these 6 proteins, namely, Rab family small GTPase, NADH dehydrogenase subunit 11, two Glutamine-rich protein 2 proteins (locus tags: 12086 and 12110), and Tigger transposable element-derived protein 1. We conducted homology modeling of these 5 proteins followed by their active site identification. These proteins were subjected to molecular docking against 105 anti-bacterial ligand compounds as potential drugs. Subsequently, the 10 best-docked compounds were determined for each protein and ranked according to the number of interactions and their binding energies. The highest binding energy was recorded for the two Glutamine-rich protein 2 proteins with different locus tags, and results have shown that the protein-inhibitor complex was stable throughout the simulation run. Moreover, future in-vitro studies could validate the findings of our in-silico analysis and identify potential therapeutic drugs against N. fowleri infections.

Crosstalk between the Rho and Rab family of small GTPases in neurodegenerative disorders.

Neurodegeneration is associated with defects in cytoskeletal dynamics and dysfunctions of the vesicular trafficking and sorting systems. In the last few decades, studies have demonstrated that the key regulators of cytoskeletal dynamics are proteins from the Rho family GTPases, meanwhile, the central hub for vesicle sorting and transport between target membranes is the Rab family of GTPases. In this regard, the role of Rho and Rab GTPases in the induction and maintenance of distinct functional and morphological neuronal domains (such as dendrites and axons) has been extensively studied. Several members belonging to these two families of proteins have been associated with many neurodegenerative disorders ranging from dementia to motor neuron degeneration. In this analysis, we attempt to present a brief review of the potential crosstalk between the Rab and Rho family members in neurodegenerative pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, and amyotrophic lateral sclerosis (ALS).

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells.

Membrane polarity, defined as the asymmetric distribution of lipids and proteins in the plasma membrane, is a critical prerequisite for the development of multicellular tissues, such as epithelia and endothelia. Membrane polarity is regulated by polarized trafficking of membrane components to specific membrane domains and requires the presence of intramembrane diffusion barriers that prevent the intermixing of asymmetrically distributed membrane components. This intramembrane diffusion barrier is localized at the tight junctions (TJs) in these cells. Both the formation of cell-cell junctions and the polarized traffic of membrane proteins and lipids are regulated by Rho and Rab family small GTPases. In this review article, we will summarize the recent developments in the regulation of apico-basal membrane polarity by polarized membrane traffic and the formation of the intramembrane diffusion barrier in epithelial cells with a particular focus on the role of Rho and Rab family small GTPases.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver

Introduction and ObjectiveAlcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol‐related death is alcohol‐associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid‐degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD‐lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well‐known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy.Materials and MethodsTo this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high‐resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD‐lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat.ResultsFirst, confocal microscopy revealed that GFP‐Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2‐fold) and LD number (1.6‐fold), and also blocked the catabolism of LDs in lipid‐loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH‐fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult.Significance/ImplicationThis study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Methods for Establishing Rab Knockout MDCK Cells.

The Rab family small GTPases are key regulators of intracellular membrane traffic that are conserved in all eukaryotic cells. Rabs are thought to regulate various steps of membrane traffic, including the budding, transport, tethering, docking, and fusion of vesicles or organelles. Approximately 60 different Rabs have been identified in mammals, and each Rab is thought to localize to a specific membrane compartment and regulate its trafficking in a timely manner. Although a few mammalian Rabs have been thoroughly studied, the precise function of the majority of them remains poorly understood. In a recent study, we established a comprehensive collection of Rab-knockout (KO) renal epithelial cells (i.e., Madin-Darby canine kidney [MDCK] II cells) by using Cas9-mediated genome editing technology to analyze the function of each Rab or closely related Rabs in cell viability (or growth), organelle morphology, and epithelial morphogenesis. In this chapter, we describe the procedures for generating Rab-KO MDCK II cells in detail.

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions.

Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

Rab GTPases in the differential processing of phagocytosed pathogens versus efferocytosed apoptotic cells.

Phagocytosis is an important feature of innate immunity in which invading microorganisms are engulfed, killed and degraded - and in some immune cells, their antigens presented to adaptive immune system. A closely related process, efferocytosis, removes apoptotic cells, and is essential for the maintenance of homeostasis. Both phagocytosis and efferocytosis are tightly regulated processes that involve target recognition and uptake through specific receptors, followed by endolysosomal trafficking and processing of the internalized target. Central to the uptake and trafficking of these targets are the Rab family of small GTPases, which coordinate the engulfment and trafficking of both phagocytosed and efferocytosed materials through the endolysosomal system. Because of this regulatory function, Rab GTPases are often targeted by pathogens to escape phagocytosis. In this review, we will discuss the shared and differential roles of Rab GTPases in phagocytosis and efferocytosis.

Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B.

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation.

RAB39B's role in membrane traffic, autophagy, and associated neuropathology.

Neuropathological disorders are increasingly associated with dysfunctions in neuronal membrane traffic and autophagy, with defects among members of the Rab family of small GTPases implicated. Mutations in the human Xq28 localized gene RAB39B have been associated with X-linked neurodevelopmental defects including macrocephaly, intellectual disability, autism spectrum disorder (ASD), as well as rare cases of early-onset Parkinson's disease (PD). Despite the finding that RAB39B regulates GluA2 trafficking and could thus influence synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit composition, reasons for the wide-ranging neuropathological consequences associated with RAB39B defects have been unclear. Recent studies have now unraveled possible mechanisms underlying the neuropathological roles of this brain-enriched small GTPase. Studies in RAB39B knockout mice showed that RAB39B interacts with components of Class I phosphatidylinositol-3-kinase (PI3K) signaling. In its absence, the PI3K-AKT-mechanistic target of rapamycin signaling pathway in neural progenitor cells (NPCs) is hyperactivated, which promotes NPC proliferation, leading to macrocephaly and ASD. Pertaining to early-onset PD, a complex of C9orf72, Smith-Magenis syndrome chromosome region candidate 8 and WD repeat domain 41 that functions in autophagy has been identified as a guanine nucleotide exchange factor of RAB39B. Here, recent findings that have shed light on our mechanistic understanding of RAB39B's role in neurodevelopmental and neurodegenerative pathologies are reviewed. Caveats and unanswered questions are also discussed, and future perspectives outlined.

Rab family of small GTPases: an updated view on their regulation and functions.

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane‐bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP‐loaded) state and an inactive (GDP‐loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post‐translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector‐recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up‐to‐date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.