Structural basis of activation of the tumor suppressor protein neurofibromin.

Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Euryarchaeota, using the model organism Haloferax volcanii. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.

Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells.

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anticancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. It also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have used the near-diploid hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with WT KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor -induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than WT cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signaling and developing effective anti-KRAS reagents with minimum cytotoxicity on WT cells.

Requirement of Rab5 GTPase during heat stress-induced endocytosis in yeast.

The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause the denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for the maintenance of PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes the mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p, the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.

Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling.

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome-mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2-GTP enters cilia by binding to IFT-B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT-B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP-locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine-tune cilia-dependent signaling for normal embryonic development and organismic homeostasis.

ARF6 plays a general role in targeting palmitoylated proteins from the Golgi to the plasma membrane.

Protein palmitoylation is a post-translational lipid modification of proteins. Accumulating evidence reveals that palmitoylation functions as a sorting signal to direct proteins to destinations; however, the sorting mechanism remains largely unknown. Here, we show that ARF6 plays a general role in targeting palmitoylated proteins from the Golgi to the plasma membrane (PM). Through shRNA screening, we identified ARF6 as the key small GTPase in targeting CD36, a palmitoylated protein, from the Golgi to the PM. We found that the N-terminal myristoylation of ARF6 is required for its binding with palmitoylated CD36, and the GTP-bound form of ARF6 facilitates the delivery of CD36 to the PM. Analysis of stable isotope labeling by amino acids in cell culture revealed that ARF6 might facilitate the sorting of 359 of the 531 palmitoylated PM proteins, indicating a general role of ARF6. Our study has thus identified a sorting mechanism for targeting palmitoylated proteins from the Golgi to the PM.

FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation.

Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

GTPase splice variants RAC1 and RAC1B display isoform-specific differences in localization, prenylation, and interaction with the chaperone protein SmgGDS.

Identifying events that regulate the prenylation and localization of small GTPases will help define new strategies for therapeutic targeting of these proteins in disorders such as cancer, cardiovascular disease, and neurological deficits. Splice variants of the chaperone protein SmgGDS (encoded by RAP1GDS1) are known to regulate prenylation and trafficking of small GTPases. The SmgGDS-607 splice variant regulates prenylation by binding preprenylated small GTPases but the effects of SmgGDS binding to the small GTPase RAC1 versus the splice variant RAC1B are not well defined. Here we report unexpected differences in the prenylation and localization of RAC1 and RAC1B and their binding to SmgGDS. Compared to RAC1, RAC1B more stably associates with SmgGDS-607, is less prenylated, and accumulates more in the nucleus. We show that the small GTPase DIRAS1 inhibits binding of RAC1 and RAC1B to SmgGDS and reduces their prenylation. These results suggest that prenylation of RAC1 and RAC1B is facilitated by binding to SmgGDS-607 but the greater retention of RAC1B by SmgGDS-607 slows RAC1B prenylation. We show that inhibiting RAC1 prenylation by mutating the CAAX motif promotes RAC1 nuclear accumulation, suggesting that differences in prenylation contribute to the different nuclear localization of RAC1 versus RAC1B. Finally, we demonstrate RAC1 and RAC1B that cannot be prenylated bind GTP in cells, indicating that prenylation is not a prerequisite for activation. We report differential expression of RAC1 and RAC1B transcripts in tissues, consistent with these two splice variants having unique functions that might arise in part from their differences in prenylation and localization.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

WDR12/RAC1 axis promoted proliferation and anti-apoptosis in colorectal cancer cells.

BACKGROUND: WD repeat domain 12 (WDR12) plays a crucial role in the ribosome biogenesis pathway. However, its biological function in colorectal cancer (CRC) remains poorly understood. Therefore, this study aims to investigate the roles of WDR12 in the occurrence and progression of CRC, as well as its underlying mechanisms. METHODS: The expression of WDR12 was assessed through The Cancer Genome Atlas (TCGA) and the Human Protein Atlas (HPA) database. Functional experiments including Celigo assay, MTT assay, and Caspase-3/7 assay were conducted to validate the role of WDR12 in the malignant progression of CRC. Additionally, mRNA chip-sequencing and ingenuity pathway analysis (IPA) were performed to identify the molecular mechanism. RESULTS: WDR12 expression was significantly upregulated in CRC tissues compared to normal colorectal tissues. Knockdown of WDR12 reduced proliferation and promoted apoptosis of CRC cell lines in vitro and in vivo experiments. Furthermore, WDR12 expression had a significantly inverse association with diseases and functions, including cancer, cell cycle, DNA replication, recombination, cellular growth, proliferation and repair, as revealed by IPA analysis of mRNA chip-sequencing data. Moreover, the activation of cell cycle checkpoint kinases proteins in the cell cycle checkpoint control signaling pathway was enriched in the WDR12 knockdown CRC cell lines. Additionally, downregulation of rac family small GTPase 1 (RAC1) occurred upon WDR12 knockdown, thereby facilitating the proliferation and anti-apoptosis of CRC cells. CONCLUSION: Our study demonstrates that the WDR12/RAC1 axis promotes tumor progression in CRC. Therefore, WDR12 may serve as a novel oncogene and a potential target for individualized therapy in CRC. These findings provide an experimental foundation for the clinical development of drugs targeting the WDR12/RAC1 axis.

Pseudorabies virus hijacks the Rab6 protein to promote viral assembly and egress.

Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.

Producing recombinant proteins in Vibrio natriegens.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Molecular basis and current insights of atypical Rho small GTPase in cancer.

Atypical Rho GTPases are a subtype of the Rho GTPase family that are involved in diverse cellular processes. The typical Rho GTPases, led by RhoA, Rac1 and Cdc42, have been well studied, while relative studies on atypical Rho GTPases are relatively still limited and have great exploration potential. With the increase in studies, current evidence suggests that atypical Rho GTPases regulate multiple biological processes and play important roles in the occurrence and development of human cancers. Therefore, this review mainly discusses the molecular basis of atypical Rho GTPases and their roles in cancer. We summarize the sequence characteristics, subcellular localization and biological functions of each atypical Rho GTPase. Moreover, we review the recent advances and potential mechanisms of atypical Rho GTPases in the development of multiple cancers. A comprehensive understanding and extensive exploration of the biological functions of atypical Rho GTPases and their molecular mechanisms in tumors will provide important insights into the pathophysiology of tumors and the development of cancer therapeutic strategies.

Expression analysis of type I ARF small GTPases ARF1-3 during mouse brain development.

BACKGROUND: ARF (ADP-ribosylation factor) GTPases are major regulators of intracellular trafficking, and classified into 3 groups (Type I - III), among which the type I group members, ARF1 and 3, are responsible genes for neurodevelopmental disorders. METHODS: In this study, we analysed the expression of Type I ARFs ARF1-3 during mouse brain development using biochemical and morphological methods. RESULTS: Western blotting analyses revealed that ARF1-3 are weakly expressed in the mouse brain at embryonic day 13 and gradually increase until postnatal day 30. ARF1-3 appear to be abundantly expressed in various telencephalon regions. Biochemical fractionation studies detected ARF1-3 in the synaptosome fraction of cortical neurons containing both pre- and post-synapses, however ARF1-3 were not observed in post-synaptic compartments. In immunohistochemical analyses, ARF1-3 appeared to be distributed in the cytoplasm and dendrites of cortical and hippocampal neurons as well as in the cerebellar molecular layer including dendrites of Purkinje cells and granule cell axons. Immunofluorescence in primary cultured hippocampal neurons revealed that ARF1-3 are diffusely distributed in the cytoplasm and dendrites with partial colocalization with a pre-synaptic marker, synaptophysin. CONCLUSIONS: Overall, our results support the notion that ARF1-3 could participate in vesicle trafficking both in the dendritic shaft (excluding spines) and axon terminals (pre-synaptic compartments).

The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB.

Both RalA and RalB interact with the ubiquitous calcium sensor, calmodulin (CaM). New structural and biophysical characterisation of these interactions strongly suggests that, in the native membrane-associated state, only RalA can be extracted from the membrane by CaM and this non-canonical interaction could underpin the divergent signalling roles of these closely related GTPases. The isoform specificity for RalA exhibited by CaM is hypothesised to contribute to the disparate signalling roles of RalA and RalB in mitochondrial dynamics. This would lead to CaM shuttling RalA to the mitochondrial membrane but leaving RalB localisation unperturbed, and in doing so triggering mitochondrial fission pathways rather than mitophagy.

Ras inhibitor CAPRI enables neutrophil-like cells to chemotax through a higher-concentration range of gradients.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.

Towards Targeting Endothelial Rap1B to Overcome Vascular Immunosuppression in Cancer.

The vascular endothelium, a specialized monolayer of endothelial cells (ECs), is crucial for maintaining vascular homeostasis by controlling the passage of substances and cells. In the tumor microenvironment, Vascular Endothelial Growth Factor A (VEGF-A) drives tumor angiogenesis, leading to endothelial anergy and vascular immunosuppression-a state where ECs resist cytotoxic CD8+ T cell infiltration, hindering immune surveillance. Immunotherapies have shown clinical promise. However, their effectiveness is significantly reduced by tumor EC anergy. Anti-angiogenic treatments aim to normalize tumor vessels and improve immune cell infiltration. Despite their potential, these therapies often cause significant systemic toxicities, necessitating new treatments. The small GTPase Rap1B emerges as a critical regulator of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) signaling in ECs. Our studies using EC-specific Rap1B knockout mice show that the absence of Rap1B impairs tumor growth, alters vessel morphology, and increases CD8+ T cell infiltration and activation. This indicates that Rap1B mediates VEGF-A's immunosuppressive effects, making it a promising target for overcoming vascular immunosuppression in cancer. Rap1B shares structural and functional similarities with RAS oncogenes. We propose that targeting Rap1B could enhance therapies' efficacy while minimizing adverse effects by reversing endothelial anergy. We briefly discuss strategies successfully developed for targeting RAS as a model for developing anti-Rap1 therapies.