Small GTPase Research Articles

BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways.

We identify BEACH domain-containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.

Spatial and planar profiling of Rac1/Cdc42 signaling in Alzheimer's disease brain.

A neuropathogenic hallmark of Alzheimer's disease (AD) is loss of neuronal synapses in selective brain regions. Small GTPases Rac1 and Cdc42, critical modulators of synaptic architecture and remodeling, are dysregulated in AD brains and are potential AD therapeutic targets. However, the exact contribution of their signaling to AD is still not clear. We intend to investigate the hypothesis that Rac1/Cdc42 activity changes in a spatial and planar dependent manner in relation to AD. We applied anti-pRac/Cdc42 (Serine 71 phosphorylation), which recognizes an inactive form of Rac1/Cdc42, and anti-pPAK (Threonine 423 phosphorylation), which detects the active PAK, a positive downstream effector of Rac1/Cdc42 signaling. For this study, triple transgenic mouse model (3xTg-AD) brain and human AD brain samples were used. pRac/Cdc42 expression was decreased in 3xTg-AD mouse cortex while pPAK expression increased compared to wild-type mouse cortex. Immunostaining of mouse brain serial sections revealed increased pRac/Cdc42 expression in the rostral region, decreased expression in the caudal region, and pPAK showed an overall opposite trend of pRac/Cdc42. There was also a brain plane specific nuclear to cytoplasmic redistribution of pRac/Cdc42. Human non-dementia and AD cortex and cerebellum showed differential expressions for pRac/Cdc42, Rac1, and Cdc42. Mouse whole transcriptome analysis demonstrated spatial dependent expression of Rac1/Cdc42 signaling-associated genes in neuronal and non-neuronal (astrocyte) populations of 3xTg-AD hippocampi. Rac1/Cdc42 signaling is dysregulated in both 3xTg-AD mouse and AD human brain and Rac1/Cdc42 activity level changes along the spatial and planar dimensions.

Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase

β-adrenergic signalling activates cAMP-dependent protein kinase (PKA), which regulates the activity of L-type voltage-gated calcium channels such as CaV1.2. Several PKA target sites in the C-terminal tail of CaV1.2 have been identified, and their phosphorylation has been suggested to increase currents in specific tissues or heterologous expression systems. However, augmentation of CaV1.2 currents in the heart is instead mediated by phosphorylation of Rad, a small GTPase that can inhibit CaV1.2. It is unclear how each of the proposed target sites in CaV1.2 and Rad rank towards their recognition by PKA, which could reveal a preferential phosphorylation. Here we used quantitative assays on three CaV1.2 and four Rad sites. Isothermal titration calorimetry (ITC) and enzyme kinetics show that there are two Tiers of targets, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming Tier 1 substrates for PKA. These share a common feature with two Arginine residues at specific positions that can anchor the peptide into the substrate binding cleft of PKA. In contrast, PKA shows minimal activity for the other, Tier 2 substrates, characterized by low kcat values and undetectable binding via ITC. The existence of two Tiers suggests that PKA regulation of the CaV1.2 complex may occur in a graded fashion. We report crystal structures of the PKA catalytic subunit with and without a CaV1.2 and test the importance of several anchoring residues via mutagenesis. Different target sites utilize different anchors, highlighting the plasticity of PKAc to recognize substrates.

Rab8a restores diverse innate functions in CD11c+CD11b+ dendritic cells from aged mice

Age-related alterations of the immune system compromise the host’s ability to respond to pathogens, but how immune aging is regulated is still poorly understood. Here, we identify via transcriptomic analysis of splenic DCs and bone marrow derived dendritic cells (BMDC) of young and aged mice, the small GTPase Rab8a as a regulator of dendritic cell (DC) functions in mice. CD11c+CD11b+ DCs of aged in comparison to young host exhibit a diminished type I IFN response upon viral stimulation and inefficiently present exogenous antigens to CD8+ T cells in vitro and in vivo. Rab8a overexpression, which is accompanied by the upregulation of Rab11, restores the functionality of these aged DCs, whereas knockdown of Rab8a reduces functionality of DCs from young mice. Mechanistically, Rab8a and Rab11 cooperate to induce efficient trafficking of peptide loaded class I MHC molecules from the ER to the cell surface. We propose that targeting Rab8a might serve as a strategy to restore DC functionality in the context of immune aging.

Phosphorylation on serine 72 modulates Rab7A palmitoylation and retromer recruitment.

Rab7A has a key role in regulating membrane trafficking at late endosomes. By interacting with several different effectors, this small GTPase controls late endosome mobility, orchestrates fusion events between late endosomes and lysosomes, and participates in the formation of and regulates the fusion between autophagosomes and lysosomes. Rab7A is also responsible for the spatiotemporal recruitment of retromer, which is required for the endosome-to-TGN retrieval of cargo-receptors such as sortilin and CI-MPR. Recently several post-translational modifications have been shown to modulate Rab7A functions, including palmitoylation, ubiquitination and phosphorylation. Here we show that phosphorylation of Rab7A at serine 72 is important to modulate its interaction with retromer, as the non-phosphorylatable Rab7AS72A mutant is not able to interact with and recruit retromer to late endosomes. We have previously shown that Rab7A palmitoylation is also required for efficient retromer recruitment. We found that palmitoylation of Rab7AS72A is reduced compared to the wild-type protein, suggesting an interplay between S72 phosphorylation and palmitoylation in regulating the Rab7A/retromer interaction. Finally, we identify NEK7 as a kinase required to phosphorylate Rab7A to promote retromer binding and recruitment.

Research progress in the small G-protein Rac1

The small G-protein Rac1 is the main regulatory factor of the actin cytoskeleton. Rac1 cycles between the inactive GDP-bound form and the active GTP-bound form. Rac1 not only promotes viral replication and infection, but also regulates the actin cytoskeleton rearrangement, adhesion, and invasion of glioma cells. In addition, Rac1 is implicated in human diseases such as tumors and epilepsy. This article reviews the latest research on the small G-protein Rac1 in virology, cell biology, and human pathology. It is found that the existence of Rac1 is closely related to the replication and infection of viruses, that is, inhibiting the existence of Rac1 can effectively reduce the replication and transportation of viruses, providing new ideas for the development of various therapeutic drugs targeting Rac1.

ARMH3 is an ARL5 effector that promotes PI4KB-catalyzed PI4P synthesis at the trans-Golgi network

ARL5 is a member of the ARF family of small GTPases that is recruited to the trans-Golgi network (TGN) by another ARF-family member, ARFRP1, in complex with the transmembrane protein SYS1. ARL5 recruits its effector, the multisubunit tethering complex GARP, to promote SNARE-dependent fusion of endosome-derived retrograde transport carriers with the TGN. To further investigate the function of ARL5, we sought to identify additional effectors. Using proximity biotinylation and protein interaction assays, we found that the armadillo-repeat protein ARMH3 (C10orf76) binds to active, but not inactive, ARL5, and that it is recruited to the TGN in a SYS1-ARFRP1-ARL5-dependent manner. Unlike GARP, ARMH3 is not required for the retrograde transport of various cargo proteins. Instead, ARMH3 functions to activate phosphatidylinositol 4-kinase IIIβ (PI4KB), accounting for the main pool of phosphatidylinositol 4-phosphate (PI4P) at the TGN. This function contributes to recruitment of the oncoprotein GOLPH3 and glycan modifications at the TGN. These studies thus identify the SYS1-ARFRP1-ARL5-ARMH3 axis as a regulator of PI4KB-dependent generation of PI4P at the TGN.

Functional dissection of prenyltransferases reveals roles in endocytosis and secretory vacuolar sorting in type 2-ME49 strain of Toxoplasma gondii

ABSTRACT Prenyltransferases act essential roles in the prenylation modification, which is significant for proteins, like small GTPases to execute various important activities in Toxoplasma gondii (T.gondii). The structures and partial functions of prenyltransferases (FTase, GGTase-I, and GGTase-II) in prenylation process have been dissected in T. gondii. However, the cellular effects of prenyltransferases on type 2-ME49 strain of Toxoplasma are largely unknown. To address this gap, CRISPR/Cas9-based gene-editing technology was employed to construct conditional knockdown strains of prenyltransferases in ME49 strain. Subsequent observation of ingestion ability of host cytosolic molecules (e.g, green fluorescent protein [GFP]) and status of secretory vacuolar sorting post-knockdown of prenyltransferases revealed significant findings. Our study demonstrated that degradation of FTase and GGTase-II notably affected the trafficking of endocytic GFP and vacuolar secretory trafficking to rhoptry bulb. Additionally, depletion of GGTase-II led to disordered endoplasmic reticulum and microtubules, as well as impaired gliding motility. The integrity of mitochondrion was damaged after degradation of GGTase-I. These findings underscore the critical functions of prenyltransferases in endocytosis and secretory vacuolar sorting in ME49 strain of T. gondii, thereby enhancing our understanding of prenyltransferases as potential drug targets.

Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation.

The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.

Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis

Melanoma arising from pigment-producing melanocytes is the deadliest form of skin cancer. Extensive ultraviolet light exposure is a major cause of melanoma and individuals with low levels of melanin are at particular risk. Humans carrying gain-of-function polymorphisms in the melanosomal/endolysosomal two-pore cation channel TPC2 present with hypopigmentation, blond hair, and albinism. Loss of TPC2 is associated with decreased cancer/melanoma proliferation, migration, invasion, tumor growth and metastasis formation, and TPC2 depleted melanoma cells show increased levels of melanin. How TPC2 activity is controlled in melanoma and the downstream molecular effects of TPC2 activation on melanoma development remain largely elusive. Here we show that the small GTPase Rab7a strongly enhances the activity of TPC2 and that effects of TPC2 on melanoma hallmarks, in vitro and in vivo strongly depend on the presence of Rab7a, which controls TPC2 activity to modulate GSK3β, β-Catenin, and MITF, a major regulator of melanoma development and progression.

R-Ras1 and R-Ras2 regulate mature oligodendrocyte subpopulations.

In the mammalian central nervous system, axonal myelination, executed by mature oligodendrocytes (MOLs), enables rapid neural transmission. Conversely, myelin deficiencies are hallmark features of multiple sclerosis, optic neuromyelitis, and some leukodystrophies. Recent studies have highlighted that MOLs are heterogeneous; however, how MOL subpopulations are specified and balanced in physiological settings is poorly understood. Previous works have demonstrated an essential role of the small GTPases R-Ras1 and R-Ras2 in the survival and myelination of oligodendrocytes. In this study, we aimed to determine how R-Ras1 and R-Ras2 contribute to the heterogeneity of MOL subpopulations. Our results evidence that R-Ras1 and R-Ras2 affect specification into the distinct subpopulations MOL1, MOL2, and MOL5/6, which in turn vary in their dependence of these GTPases. In R-Ras1 and/or R-Ras2 mutant mice, we observed an increase in the MOL1 subpopulation and a decrease in the MOL2 and MOL5/6 subpopulations. We identified R-Ras1 and R-Ras2 as key elements in balancing the heterogeneity of MOLs. Our results contribute to the understanding of the molecular mechanisms underlying the heterogeneity of MOLs and the myelination processes, which is crucial for innovating regenerative therapies for nervous system disorders.

Heterogeneous folding landscapes and predetermined breaking points within a protein family.

The accurate prediction of protein structures with artificial intelligence has been a spectacular success. Yet, how proteins fold into their native structures inside the cell remains incompletely understood. Of particular interest is to rationalize how proteins interact with the protein homeostasis network, an organism specific set of protein folding and quality control enzymes. Failure of protein homeostasis leads to widespread misfolding and aggregation, and thus neurodegeneration. Here, I present a comparative analysis of the folding of 16 single-domain proteins from the same organism across a protein family, the Saccharomyces cerevisiae small GTPases. Using computational modeling to directly probe protein folding dynamics, this work shows how near identical structures from the same folding environment can exhibit heterogeneous folding landscapes. Remarkably, yeast small GTPases are found to unfold along different pathways either via the N- or C-terminus initiated by structure-encoded predetermined breaking points. Degrons as recognition signals for ubiquitin-dependent degradation were systematically absent from the initial unfolding sites, as if to protect from too rapid degradation upon spontaneous unfolding or before completion of the folding. The presented results highlight a direct coordination of folding pathway and protein homeostasis interaction signals across a protein family. A deeper understanding of the interdependence of proteins with their folding environment will help to rationalize and combat diseases linked to protein misfolding and dysregulation. More generally, this work underlines the importance of understanding protein folding in the cellular context, and highlights valuable constraints towards a systems-level understanding of protein homeostasis.

Small ARF-like 2 GTPase TITAN 5 is linked with the dynamic regulation of IRON-REGULATED TRANSPORTER 1.

Iron acquisition is crucial for plants. The abundance of IRON-REGULATED TRANSPORTER 1 (IRT1) is controlled through endomembrane trafficking, a process that requires small ARF-like GTPases. Only few components are known that are involved in the vesicular trafficking of specific cargo. Here, we report that the ARF-like GTPase TITAN 5 (TTN5) interacts with the large cytoplasmic variable region and protein-regulatory platform of IRT1. Heterozygous ttn5-1 plants can display a reduced activity of root iron reductase. This activity is needed for iron uptake via IRT1. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at places, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. TTN5 can also interact with peripheral membrane proteins that are components of the IRT1 regulation machinery, like the trafficking factor SNX1, the C2 domain protein EHB1 and the SEC14-GOLD protein PATL2. Hence, the link between iron acquisition and vesicular trafficking involving a small GTPase of the ARF family opens up the possibility to study the involvement of TTN5 in nutritional cell biology in the endomembrane system.

Quantitative trait locus mapping and OsFLOq12 identification for rice grain hardness: towards improved rice flour for wheat substitution.

Recent shifts in consumer dietary preferences have led to a significant decline in rice consumption in Korea, resulting in surplus rice production. To address this issue, rice flour has been proposed as a substitute for wheat flour. However, the physical, chemical and structural differences between rice and wheat, particularly in grain hardness, pose challenges in using rice flour as an alternative. Understanding the genetic factors that influence rice grain hardness is crucial for improving the milling process and producing high-quality rice flour suitable for wheat flour substitution. In this study, various grain traits, including length, width, thickness, length-to-width ratio and hardness, were measured in a population of brown and milled rice. Quantitative trait locus (QTL) analysis revealed a significant association between grain hardness and thickness, with QTLs for grain hardness mapped on chromosomes 1 and 12 for brown and milled rice, respectively. A total of 20 candidate genes related to grain hardness were identified through QTL analysis. Among them, OsFLOq12 (LOC_Os12g43550) was identified as a key gene influencing grain hardness, which encodes a Ras small GTPase. Phenotypic analysis showed differences in endosperm appearance and particle size between lines with low and high grain hardness. The genetic analysis of OsFLOq12 revealed a single nucleotide polymorphism associated with grain hardness. This study provides valuable insights into the genetic background of grain hardness, offering a foundation for breeding rice varieties optimized for flour production as a viable substitute for wheat flour. © 2024 Society of Chemical Industry.

Abstract PR012: A first-in-class EGFR-directed KRAS G12V selective inhibitor

Abstract The KRAS proto-oncogene encodes a small GTPase that is crucial for the activation of intracellular signaling pathways that control cell proliferation, survival and differentiation. KRAS is frequently mutated in cancer resulting in its constitutive activation and dysregulation of downstream signaling pathways that drive oncogenic transformation. KRAS G12V is the second most common KRAS mutation in cancer, and occurs frequently in lung, colon and pancreatic cancers. However, while significant advancements have been made in developing KRAS G12C, G12D and pan-KRAS inhibitors, there remain no direct KRAS G12V inhibitors in the clinic. Due to challenges targeting oncogenes with traditional small molecule approaches, RNA interference (RNAi) has become an attractive therapeutic approach for many oncogenes. RNAi has faced many obstacles as an effective cancer therapeutics, including the lack of cancer- specific tissue targeting, rapid oligonucleotide nuclease degradation and clearance from circulation, the need for endosomal escape, and unwanted immune stimulation. However, recently, the use of targetable ligands conjugated to chemically modified siRNAs have shown promise in circumventing these barriers. Here, we describe the development of EFTX-G12V, a fully-modified, mutant-selective siRNA conjugated to an EGFR ligand that shows improved tumor delivery and significant anti-tumor activity in lung and colon cancer models. We used a structure-activity relationship screening approach to identify a highly mutant-selective fully chemically modified siRNA that inhibits KRAS G12V expression at both the mRNA and protein level while completely sparing KRAS WT. Furthermore, this siRNA inhibited cancer cell growth in vitro and showed no off-target effects. To address the issue of tumor-specific delivery, we conjugated a non-mitogenic EGFR ligand to the siRNA and found enhanced delivery to tumors with varying levels of EGFR expression. EFTX-G12V significantly inhibited KRAS G12V mRNA and protein in vivo and showed significant anti-tumor activity in lung and colon xenograft models. Importantly, EFTX-G12V showed no off-target effects on WT KRAS in somatic tissues including kidney, skin and bladder. Unexpectedly, we found that mutant-selective targeting showed improved efficacy in comparison to a pan-KRAS siRNA. EFTX-G12V significantly inhibited tumor angiogenesis while pan-KRAS targeting had inconsistent effects suggesting that mutant-selective targeting has advantages within the tumor microenvironment. Together our findings represent a technologic advance in ligand-directed, mutant-selective oncogene targeting using RNAi and reveal new biologic insights in KRAS targeting that may have broader implications with regards to safety and efficacy. Citation Format: Lyla Stanland, Hayden Huggins, Alessandro Porrello, Yogitha Chareddy, Salma Azam, Jillian Perry, Pradeep Pallan, Kristina Whately, Lincy Edatt, Matthew Fleming, Jonah Im, Christina Gutierrez-Ford, Imani Simmons, Vandanaa Jayaprakash, Rani Sellers, Gabriela de la Cruz, Albert Wielgus, Martin Egli, Albert Bowers, Chad Pecot. A first-in-class EGFR-directed KRAS G12V selective inhibitor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR012.

RhoA Allosterically Activates Phospholipase Cε via its EF Hands.

Phospholipase Cε (PLCε) cleaves phosphatidylinositol lipids to increase intracellular Ca 2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLCε is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLCε to initiate a cardioprotective pathway, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLCε. The G protein binds a unique insertion in the PLCε EF hands. Deletion of or mutations to this PLCε insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLCε allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.

EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasite Entamoeba histolytica.

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration.

FRET verification of crucial interaction sites in RhoA regulation mediated by RhoGDI

The small GTPase Rho family are the major factors in mediating actin cytoskeleton dynamics. Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) serve as important negative regulators by complexing with inactive Rho into the cytoplasm. However, how these two molecules interact still needs experimental verification. Based on fluorescence resonance energy transfer (FRET) measurements, we would demonstrate crucial sites in RhoGDI and RhoA for this regulatory role. Cotransfection of RhoGDI markedly reduced RhoA or Cdc42 activity in airway smooth muscle (ASM) cells, while D185R-RhoGDI mutant reversed this decrease, indicating that RhoGDI Asp185 residue is essential for the molecular interaction. R68D-RhoA (mutation in the switch II region) resulted in a deficiency in RhoGDI regulation, while TV37/38NG-RhoA (in the switch I region) displayed low RhoA activity. Hence, the Arg68 site in RhoA is indispensable for regulation by RhoGDI, and Thr37Val38 site is important for maintaining RhoA activity. Additionally, microtubule but not actin cytoskeleton showed inhibitory role in RhoA activity, while the dissolution of either cytoskeleton did not change the regulatory role of RhoGDI. In checking the downstream effect, reduction of RhoA activity induced by PDGF stimulation or RhoGDI decreased cellular stress fibers. In this study, FRET visualization was applied to have experimentally demonstrated the interaction sites and crucial role of RhoGDI in regulating RhoA activity.Graphical

Murine colon cancer derived cells exhibit heterogeneous resistance profiles against an oncolytic virus

Oncolytic virotherapy has shown efficacy in various animal models and a few human cancers. However, there are still significant limitations for the implementation of these therapies. One such limitation is the emergence of cellular resistances, which may appear rapidly considering the high genetic heterogeneity of most tumors. We previously showed that cellular resistance to an oncolytic virus can be mediated by the chronic activation of innate immunity. Here, we explored the existence of additional resistance mechanisms in murine colon cancer-derived cells. For this purpose, we isolated two cellular clones that were resistant to the oncolytic virus VSV-D51. While one of the clones showed a strong resistance profile associated with increased cytokine-mediated antiviral responses, the other clone showed a lower level of resistance that involves cytoskeletal reorganization, signaling by small GTPases, and cell structural changes. These results demonstrate the capacity of tumor cells to deploy heterogeneous mechanisms of resistance to oncolytic viruses.

Rab40 GTPases regulate AMBRA1-mediated transcription and cell migration.

The Rab40 subfamily are unique small monomeric GTPases that form CRL5-based ubiquitin E3 ligase complex and regulate ubiquitylation of specific target proteins. Recent studies have shown that Rab40s play an important role in regulating cell migration, but the underlying mechanisms of Rab40/CRL5 complex function are still not fully understood. In this study we identified AMBRA1 as a novel binding partner of Rab40 GTPases and showed that this interaction mediates a bi-directional crosstalk between CRL4 and CRL5 E3 ligases. Importantly, we found that Rab40/CRL5 ubiquitylates AMBRA1, which does not result in AMBRA1 degradation, but instead it seems to induce AMBRA1-dependent regulation of gene transcription. The global transcriptional profiles identified by RNA-seq showed that AMBRA1 regulates transcription of genes related to cell adhesion and migration. Additionally, we have shown that AMBRA1-dependent transcription regulation does not require the enzymatic activity of AMBRA1/CRL4, and that Rab40-induced AMBRA1 ubiquitylation leads to dissociation of AMBRA1/CRL4 complex. Taken together, our findings reveal a novel function of Rab40/CRL5 complex as an important regulator for AMBRA1-dependent transcription of genes involved in cell migration.