X-Linked Epilepsies: A Narrative Review.

X-linked epilepsies are a heterogeneous group of epileptic conditions, which often overlap with X-linked intellectual disability. To date, various X-linked genes responsible for epilepsy syndromes and/or developmental and epileptic encephalopathies have been recognized. The electro-clinical phenotype is well described for some genes in which epilepsy represents the core symptom, while less phenotypic details have been reported for other recently identified genes. In this review, we comprehensively describe the main features of both X-linked epileptic syndromes thoroughly characterized to date (PCDH19-related DEE, CDKL5-related DEE, MECP2-related disorders), forms of epilepsy related to X-linked neuronal migration disorders (e.g., ARX, DCX, FLNA) and DEEs associated with recently recognized genes (e.g., SLC9A6, SLC35A2, SYN1, ARHGEF9, ATP6AP2, IQSEC2, NEXMIF, PIGA, ALG13, FGF13, GRIA3, SMC1A). It is often difficult to suspect an X-linked mode of transmission in an epilepsy syndrome. Indeed, different models of X-linked inheritance and modifying factors, including epigenetic regulation and X-chromosome inactivation in females, may further complicate genotype-phenotype correlations. The purpose of this work is to provide an extensive and updated narrative review of X-linked epilepsies. This review could support clinicians in the genetic diagnosis and treatment of patients with epilepsy featuring X-linked inheritance.

ZO-1 Regulates Hippo-Independent YAP Activity and Cell Proliferation via a GEF-H1- and TBK1-Regulated Signalling Network.

Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, induced loss of cell density-dependent proliferation control, and promoted apoptosis and necrosis. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on two transcriptional regulators: YAP and ZONAB. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were found to cooperate to activate YAP/TEAD in ZO-1-deficient cells. Thus, ZO-1 controled cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.

PREX2 contributes to radiation resistance by inhibiting radiotherapy-induced tumor immunogenicity via cGAS/STING/IFNs pathway in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) lacks established biomarkers or molecular targets for predicting or enhancing radiation response. Phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 2 (PREX2) exhibits intricate implications in tumorigenesis and progression. Nevertheless, the precise role and underlying mechanisms of PREX2 in CRC radioresistance remain unclear. METHODS: RNA-seq was employed to identify differentially expressed genes between radioresistant CRC cell lines and their parental counterparts. PREX2 expression was scrutinized using Western blotting, real-time PCR, and immunohistochemistry. The radioresistant role of PREX2 was assessed through in vitro colony formation assay, apoptosis assay, comet assay, and in vivo xenograft tumor models. The mechanism of PREX2 was elucidated using RNA-seq and Western blotting. Finally, a PREX2 small-molecule inhibitor, designated PREX-in1, was utilized to enhance the efficacy of ionizing radiation (IR) therapy in CRC mouse models. RESULTS: PREX2 emerged as the most significantly upregulated gene in radioresistant CRC cells. It augmented the radioresistant capacity of CRC cells and demonstrated potential as a marker for predicting radioresistance efficacy. Mechanistically, PREX2 facilitated DNA repair by upregulating DNA-PKcs, suppressing radiation-induced immunogenic cell death, and impeding CD8+ T cell infiltration through the cGAS/STING/IFNs pathway. In vivo, the blockade of PREX2 heightened the efficacy of IR therapy. CONCLUSIONS: PREX2 assumes a pivotal role in CRC radiation resistance by inhibiting the cGAS/STING/IFNs pathway, presenting itself as a potential radioresistant biomarker and therapeutic target for effectively overcoming radioresistance in CRC.

Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

Real-Time Monitoring of RAS Activity Using In Vitro and In-Cell NMR Spectroscopy.

The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.

Targeting RCC1 to block the human soft-tissue sarcoma by disrupting nucleo-cytoplasmic trafficking of Skp2.

Soft-tissue sarcomas (STS) emerges as formidable challenges in clinics due to the complex genetic heterogeneity, high rates of local recurrence and metastasis. Exploring specific targets and biomarkers would benefit the prognosis and treatment of STS. Here, we identified RCC1, a guanine-nucleotide exchange factor for Ran, as an oncogene and a potential intervention target in STS. Bioinformatics analysis indicated that RCC1 is highly expressed and correlated with poor prognosis in STS. Functional studies showed that RCC1 knockdown significantly inhibited the cell cycle transition, proliferation and migration of STS cells in vitro, and the growth of STS xenografts in mice. Mechanistically, we identified Skp2 as a downstream target of RCC1 in STS. Loss of RCC1 substantially diminished Skp2 abundance by compromising its protein stability, resulting in the upregulation of p27Kip1 and G1/S transition arrest. Specifically, RCC1 might facilitate the nucleo-cytoplasmic trafficking of Skp2 via direct interaction. As a result, the cytoplasmic retention of Skp2 would further protect it from ubiquitination and degradation. Notably, recovery of Skp2 expression largely reversed the phenotypes induced by RCC1 knockdown in STS cells. Collectively, this study unveils a novel RCC1-Skp2-p27Kip1 axis in STS oncogenesis, which holds promise for improving prognosis and treatment of this formidable malignancy.

Mechanistic insights into G-protein activation via phosphorylation mediated non-canonical pathway.

Activation of heterotrimeric G-proteins (Gαßγ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of Gα that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce Gα activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gαi3, a subtype of Gα-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. In addition to weakened salt bridges at the domain interface, phosphorylations induced the unfolding of helix αF, which contributed to increased flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gαi3. Although the larger domain separation in the phosphorylated system provided an unobstructed path for the nucleotide, the accelerated release of GDP was attributed to increased fluctuations in several conserved regions like P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

MADD regulates natural killer cell degranulation through Rab27a activation.

Natural killer (NK) cells have the ability to lyse other cells through the release of lytic granules (LGs). This is in part mediated by the small GTPase Rab27a, which was first identified to play a crucial role in degranulation through the study of individuals harboring mutations in the gene encoding Rab27a. However, the guanine nucleotide exchange factor (GEF) regulating the activation of Rab27a in cytotoxic lymphocytes was unknown. Here, we show that knockout of MADD significantly decreased the levels of GTP-bound Rab27a in both resting and stimulated NK cells, and MADD-deficient NK cells and CD8+ T cells displayed severely reduced degranulation and cytolytic ability, similar to that seen with Rab27a deficiency. Although MADD colocalized with Rab27a on LGs and was enriched at the cytolytic synapse, the loss of MADD did not impact Rab27a association with LGs nor their recruitment to the cytolytic synapse. Together, our results demonstrate an important role for MADD in cytotoxic lymphocyte killing.

Cryo-EM of the Nucleosome Core Particle Bound to Ran-RCC1 Reveals a Dynamic Complex.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder-order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer.

AIMS: Exchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. MAIN METHODS: We employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. KEY FINDINGS: Inhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. CONCLUSIONS: Our findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Sustained effects on immune cell subsets and autoreactivity in multiple sclerosis patients treated with oral cladribine.

Introduction: Cladribine tablet therapy is an efficacious treatment for multiple sclerosis (MS). Recently, we showed that one year after the initiation of cladribine treatment, T and B cell crosstalk was impaired, reducing potentially pathogenic effector functions along with a specific reduction of autoreactivity to RAS guanyl releasing protein 2 (RASGRP2). In the present study we conducted a longitudinal analysis of the effect of cladribine treatment in patients with RRMS, focusing on the extent to which the effects observed on T and B cell subsets and autoreactivity after one year of treatment are maintained, modulated, or amplified during the second year of treatment. Methods: In this case-control exploratory study, frequencies and absolute counts of peripheral T and B cell subsets and B cell cytokine production from untreated patients with relapsing-remitting MS (RRMS) and patients treated with cladribine for 52 (W52), 60 (W60), 72 (W72) and 96 (W96) weeks, were measured using flow cytometry. Autoreactivity was assessed using a FluoroSpot assay. Results: We found a substantial reduction in circulating memory B cells and proinflammatory B cell responses. Furthermore, we observed reduced T cell responses to autoantigens possibly presented by B cells (RASGRP2 and a-B crystallin (CRYAB)) at W52 and W96 and a further reduction in responses to the myelin antigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) after 96 weeks. Conclusion: We conclude that the effects of cladribine observed after year one are maintained and, for some effects, even increased two years after the initiation of a full course of treatment with cladribine tablets.

De novo variants in DENND5B cause a neurodevelopmental disorder.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Single-cell analysis with childhood and adult systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE), a heterogeneous and chronic autoimmune disease, exhibit unique changes in the complex composition and transcriptional signatures of peripheral blood mononuclear cells (PBMCs). While the mechanism of pathogenesis for both childhood-onset SLE (cSLE) and adult-onset SLE (aSLE) remains unclear, cSLE patients are considered more unpredictable and dangerous than aSLE patients. In this study, we analysed single-cell RNA sequencing data (scRNA-seq) to profile the PBMC clusters of cSLE/aSLE patients and matched healthy donors and compared the PBMC composition and transcriptional variations between the two groups. Our analysis revealed that the PBMC composition and transcriptional variations in cSLE patients were similar to those in aSLE patients. Comparative single-cell transcriptome analysis between healthy donors and SLE patients revealed IFITM3, ISG15, IFI16 and LY6E as potential therapeutic targets for both aSLE and cSLE patients. Additionally, we observed that the percentage of pre-B cells (CD34-) was increased in cSLE patients, while the percentage of neutrophil cells was upregulated in aSLE patients. Notably, we found decreased expression of TPM2 in cSLE patients, and similarly, TMEM150B, IQSEC2, CHN2, LRP8 and USP46 were significantly downregulated in neutrophil cells from aSLE patients. Overall, our study highlights the differences in complex PBMC composition and transcriptional profiles between cSLE and aSLE patients, providing potential biomarkers that could aid in diagnosing SLE.

PREX1 improves homeostatic proliferation to maintain a naive CD4+ T cell compartment in older age.

The human adult immune system maintains normal T cell counts and compensates for T cell loss throughout life, mainly through peripheral homeostatic proliferation after the ability of the thymus to generate new T cells has rapidly declined at adolescence. This process is mainly driven by STAT5-activating cytokines, most importantly IL-7, and is very effective in maintaining a large naive CD4+ T cell compartment into older age. Here, we describe that naive CD4+ T cells undergo adaptations to optimize IL-7 responses by upregulating the guanine-nucleotide exchange factor PREX1 in older age. PREX1 promotes nuclear translocation of phosphorylated STAT5, thereby supporting homeostatic proliferation in response to IL-7. Through the same mechanism, increased expression of PREX1 also biases naive cells to differentiate into effector T cells. These findings are consistent with the concept that primarily beneficial adaptations during aging, i.e., improved homeostasis, account for unfavorable functions of the aged immune system, in this case biased differentiation.

TIAM-1 regulates polarized protrusions during dorsal intercalation in the Caenorhabditis elegans embryo through both its GEF and N-terminal domains.

Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the Caenorhabditis elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and thus is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterized the role of the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1 in regulating actin-based protrusive dynamics during dorsal intercalation. We found that TIAM-1 can promote formation of the main medial lamellipodial protrusion extended by intercalating cells through its canonical GEF function, whereas its N-terminal domains function to negatively regulate the generation of ectopic filiform protrusions around the periphery of intercalating cells. We also show that the guidance receptor UNC-5 inhibits these ectopic filiform protrusions in dorsal epidermal cells and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed rearrangement of epithelial cells in animal embryos.

Guanine nucleotide exchange factor RABGEF1 facilitates TNF-induced necroptosis by targeting cIAP1.

Necroptosis is a form of regulated cell death that depends on the receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). The molecular mechanisms underlying distinct instances of necroptosis have only recently begun to emerge. In the present study, we characterized RABGEF1 as a positive regulator of RIPK1/RIPK3 activation in vitro. Based on the overexpression and knockdown experiments, we determined that RABGEF1 accelerated the phosphorylation of RIPK1 and promoted necrosome formation in L929 cells. The pro-necrotic effect of RABGEF1 is associated with its E3 ubiquitin ligase activity and guanine nucleotide exchange factor (GEF) activity. We further confirmed that RABGEF1 interacts with cIAP1 protein by inhibiting its function and plays a regulatory role in necroptosis, which can be abolished by treatment with the antagonist Smac mimetic (SM)-164. In conclusion, our study highlights a potential and novel role of RABGEF1 in promoting TNF-induced cell necrosis.

HTATIP2 Overexpression was Associated With a Good Prognosis in Gastric Cancer.

Introduction: The purpose of this study was to compare the transcriptomes of poorly cohesive carcinoma (PCC; diffuse-type) and well-differentiated tubular adenocarcinoma (WD; intestinal-type) using gastric cancer (GC) tissues and cell lines and to evaluate the prognostic role of HIV-1 Tat Interactive Protein 2 (HTATIP2). Materials and Methods: We performed next-generation sequencing with 8 GC surgical samples (5 WD and 3 PCC) and 3 GC cell lines (1 WD: MKN74, and 2 PCC: KATOIII and SNU601). Immunohistochemistry was used to validate HTATIP2 expression. We performed functional analysis by HTATIP2 overexpression (OE). Kaplan-Meier survival plots and the PrognoScan database were used for survival analysis. Results: The genes with significantly reduced expression in PCC versus WD (in both tissues and cell lines) were HTATIP2, ESRP1, GRHL2, ARHGEF16, CKAP2L, and ZNF724. According to immunohistochemical staining, the HTATIP2-OE group had significantly higher number of patients with early GC (EGC) (T1) (P = .024), less lymph node (LN) metastasis (P = .008), and low TNMA stage (P = .017) than HTATIP2 underexpression (UE) group. Better survival rates were confirmed in the HTATIP2 OE group by Kaplan-Meir survival and PrognoScan analysis. In vitro, HTATIP2-OE in KATO III cells caused a significant decrease in cancer cell migration and invasion. Decreased Snail and Slug expression in HTATIP2 OE cells suggested that epithelial-mesenchymal transition is involved in this process. Conclusion: HTATIP2 might be a good prognostic marker and a candidate target for GC treatment.

Implication of Rac1 GTPase in molecular and cellular mitochondrial functions.

Rac1 is a member of the Rho GTPase family which plays major roles in cell mobility, polarity and migration, as a fundamental regulator of actin cytoskeleton. Signal transduction by Rac1 occurs through interaction with multiple effector proteins, and its activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The small protein is mainly anchored to the inner side of the plasma membrane but it can be found in endocellular compartments, notably endosomes and cell nuclei. The protein localizes also into mitochondria where it contributes to the regulation of mitochondrial dynamics, including both mitobiogenesis and mitophagy, in addition to signaling processes via different protein partners, such as the proapoptotic protein Bcl-2 and chaperone sigma-1 receptor (σ-1R). The mitochondrial form of Rac1 (mtRac1) has been understudied thus far, but it is as essential as the nuclear or plasma membrane forms, via its implication in regulation of oxidative stress and DNA damages. Rac1 is subject to diverse post-translational modifications, notably to a geranylgeranylation which contributes importantly to its mitochondrial import and its anchorage to mitochondrial membranes. In addition, Rac1 contributes to the mitochondrial translocation of other proteins, such as p53. The mitochondrial localization and functions of Rac1 are discussed here, notably in the context of human diseases such as cancers. Inhibitors of Rac1 have been identified (NSC-23766, EHT-1864) and some are being developed for the treatment of cancer (MBQ-167) or central nervous system diseases (JK-50561). Their effects on mtRac1 warrant further investigations. An overview of mtRac1 is provided here.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).