Analysis of the Guanine Nucleotide-Bound State of KRAS by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography.

Guanine nucleotides can be quantitatively analyzed by high-performance liquid chromatography (HPLC). Here we describe an ion-pair reversed-phase HPLC (IP-RP-HPLC)-based method, which enables analyzing GDP and GTP bound to small GTPases immunoprecipitated from cells. The activation status of FLAG-KRAS expressed in HEK293T cells can be investigated with the IP-RP-HPLC method. This method also can be adapted to determine the effects of compounds such as the KRAS/G12C inhibitor sotorasib on the activation status of FLAG-KRAS in the cells.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Functional Conservation of the Small GTPase Rho5/Rac1-A Tale of Yeast and Men.

Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer.

BACKGROUND: Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of T cells. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). However, the mechanism of intracellular trafficking and plasma membrane presentation of PD-1 remains unclear. METHODS: Multiple databases of lung cancer patients were integratively analyzed to screen Rab proteins and potential immune-related signaling pathways. Imaging and various biochemical assays were performed in Jurkat T cells, splenocytes, and human peripheral blood mononuclear cells (PBMCs). Rab37 knockout mice and specimens of lung cancer patients were used to validate the concept. RESULTS: Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37 small GTPase to sustain T cell exhaustion, thereby leading to poor patient outcome. PD-1 colocalized with Rab37-specific vesicles of T cells in a GTP-dependent manner whereby Rab37 mediated dynamic trafficking and membrane presentation of PD-1. However, glycosylation mutant PD-1 delayed cargo recruitment to the Rab37 vesicles, thus stalling membrane presentation. Notably, T cell proliferation and activity were upregulated in tumor-infiltrating T cells from the tumor-bearing Rab37 knockout mice compared to those from wild type. Clinically, the multiplex immunofluorescence-immunohistochemical assay indicated that patients with high Rab37+/PD-1+/TIM3+/CD8+ tumor infiltrating T cell profile correlated with advanced tumor stages and poor overall survival. Moreover, human PBMCs from patients demonstrated high expression of Rab37, which positively correlated with elevated levels of PD-1+ and TIM3+ in CD8+ T cells exhibiting reduced tumoricidal activity. CONCLUSIONS: Our results provide the first evidence that Rab37 small GTPase mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion, and the tumor promoting function of Rab37/PD-1 axis in T cells of TME in lung cancer. The expression profile of Rab37high/PD-1high/TIM3high in tumor-infiltrating CD8+ T cells is a biomarker for poor prognosis in lung cancer patients.

Systematic analysis of genotype-phenotype variability in siblings with Aicardi Goutières Syndrome (AGS).

OBJECTIVE: Aicardi Goutières Syndrome (AGS) is a genetic interferonopathy associated with multisystemic heterogeneous disease and neurologic dysfunction. AGS includes a broad phenotypic spectrum which is only partially explained by genotype. To better characterize this variability, we will perform a systematic analysis of phenotypic variability in familial cases of AGS. METHODS: Among thirteen families, twenty-six siblings diagnosed with AGS were identified from the Myelin Disorders and Biorepository Project (MDBP) at the Children's Hospital of Philadelphia. Data were collected on the age of onset, genotype, neurologic impairment, and systemic complications. Neurologic impairment was assessed by a disease-specific scale (AGS Severity Scale) at the last available clinical encounter (range: 0-11 representing severe - attenuated phenotypes). The concordance of clinical severity within sibling pairs was categorized based on the difference in AGS Scale (discordant defined as >2-unit difference). The severity classifications were compared between sibling sets and by genotype. RESULTS: Five genotypes were represented: TREX1 (n = 4 subjects), RNASEH2B (n = 8), SAMHD1 (n = 8) ADAR1 (n = 4), and IFIH1 (n = 2). The older sibling was diagnosed later relative to the younger affected sibling (median age 7.32 years [IQR = 14.1] compared to 1.54 years [IQR = 10.3]). Common presenting neurologic symptoms were tone abnormalities (n = 10/26) and gross motor dysfunction (n = 9/26). Common early systemic complications included dysphagia and chilblains. The overall cohort median AGS severity score at the last encounter was 8, while subjects presenting with symptoms before one year had a median score of 5. The TREX1 cohort presented at the youngest age and with the most severe phenotype on average. AGS scores were discordant for 5 of 13 sibling pairs, most commonly in the SAMHD1 pairs. Microcephaly, feeding tube placement, seizures and earlier onset sibling were associated with lower AGS scores (respectively, Wilcoxon rank sum: p = 0.0001, p < 0.0001, p = 0.0426, and Wilcoxon signed rank: p = 0.0239). CONCLUSIONS: In this systematic analysis of phenotypic variability in familial cases, we found discordance between siblings affected by AGS. Our results underscore the heterogeneity of AGS and suggest factors beyond AGS genotype may affect phenotype. Understanding the critical variables associated with disease onset and severity can guide future therapeutic interventions and clinical monitoring. This report reinforces the need for further studies to uncover potential factors to better understand this phenotypic variability, and consequently identify potential targets for interventions in attempt to change the natural history of the disease.

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.

TRAF4-Mediated LAMTOR1 Ubiquitination Promotes mTORC1 Activation and Inhibits the Inflammation-Induced Colorectal Cancer Progression.

Mechanistic target of rapamycin complex 1 (mTORC1) is a conserved serine/threonine kinase that integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activation requires tethering to lysosomes by the Ragulator-Rag complex. However, the dynamic regulation of the interaction between Ragulator and Rag guanosine triphosphatase (GTPase) remains unclear. In this study, that LAMTOR1, an essential component of Ragulator, is dynamically ubiquitinated depending on amino acid abundance is reported. It is found that the E3 ligase TRAF4 directly interacts with LAMTOR1 and catalyzes the K63-linked polyubiquitination of LAMTOR1 at K151. Ubiquitination of LAMTOR1 by TRAF4 promoted its binding to Rag GTPases and enhanced mTORC1 activation, K151R knock-in or TRAF4 knock-out blocks amino acid-induced mTORC1 activation and accelerates the development of inflammation-induced colon cancer. This study revealed that TRAF4-mediated LAMTOR1 ubiquitination is a regulatory mechanism for mTORC1 activation and provides a therapeutic target for diseases involving mTORC1 dysregulation.

The RRAS2 pathogenic variant (c.67G>T; p. Gly23Cys) produces Noonan syndrome with embryonal rhabdomyosarcoma.

BACKGROUND: Noonan syndrome (NS) due to the RRAS2 gene, the pathogenic variant is an extremely rare RASopathies. Our objective was to identify the potential site of RRAS2, combined with the literature review, to find the correlation between clinical phenotype and genotype. De novo missense mutations affect different aspects of the RRAS2 function, leading to hyperactivation of the RAS-MAPK signaling cascade. METHODS: Conventional G-banding was used to analyze the chromosome karyotype of the patient. Copy number variation sequencing (CNV-seq) was used to detect the chromosomal gene microstructure of the patient and her parents. The exomes of the patient and her parents were sequenced using trio-based whole exome sequencing (trio-WES) technology. The candidate variant was verified by Sanger sequencing. The pathogenicity of the variant was predicted with a variety of bioinformatics tools. RESULTS: Chromosome analysis of the proband revealed 46, XX, and no abnormality was found by CNV-seq. After sequencing and bioinformatics filtering, the variant of RRAS2(c.67G>T; p. Gly23Cys) was found in the proband, while the mutation was absent in her parents. To the best of our knowledge, our patient was with the typical Noonan syndrome, such as short stature, facial dysmorphism, and developmental delay. Furthermore, our study is the first case of NS with embryonal rhabdomyosarcoma (ERMS) caused by the RRAS2 gene mutation reported in China. CONCLUSIONS: Our investigations suggested that the heterozygous missense of RRAS2 may be a potential causal variant in a rare cause of Noonan syndrome, expanding our understanding of the causally relevant mutations for this disorder.

Reprogramming nucleolar size by genetic perturbation of the extranuclear Rab GTPases Ypt6 and Ypt32.

Reprogramming organelle size has been proposed as a potential therapeutic approach. However, there have been few reports of nucleolar size reprogramming. We addressed this question in Saccharomyces cerevisiae by studying mutants having opposite effects on the nucleolar size. Mutations in genes involved in nuclear functions (KAR3, CIN8, and PRP45) led to enlarged nuclei/nucleoli, whereas mutations in secretory pathway family genes, namely the Rab-GTPases YPT6 and YPT32, reduced nucleolar size. When combined with mutations leading to enlarged nuclei/nucleoli, the YPT6 or YPT32 mutants can effectively reprogram the nuclear/nucleolar size almost back to normal. Our results further indicate that null mutation of YPT6 causes secretory stress that indirectly influences nuclear localization of Maf1, the negative regulator of RNA Polymerase III, which might reduce the nucleolar size by inhibiting nucleolar transcript enrichment.

Small G-Protein Rheb Gates Mammalian Target of Rapamycin Signaling to Regulate Morphine Tolerance in Mice.

BACKGROUND: Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on µ-opioid receptors and downstream of the phosphatidylinositol 3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. The hypothesis was that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. METHODS: Male and female wild-type C57BL/6J mice or transgenic mice (6 to 8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for 5 consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After 5 days, the spine was harvested for Western blot or immunofluorescence analysis. RESULTS: Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27 ± 0.195-fold (P = 0.0036, n = 6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65 ± 1.10 s (P < 0.0001, n = 7) in Rheb knock-in mice compared to 10 s in control mice (10 ± 0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (maximum possible effect, 52.60 ± 9.56% in the morphine + rapamycin group vs. 16.60 ± 8.54% in the morphine group; P < 0.0001). Moreover, activation of endogenous adenosine 5'-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (maximum possible effect, 39.51 ± 7.40% in morphine + metformin group vs. 15.58 ± 5.79% in morphine group; P < 0.0001). CONCLUSIONS: This study suggests spinal Rheb as a key molecular factor for regulating mammalian target of rapamycin signaling.

Structural insights into the complex of oncogenic KRas4BG12V and Rgl2, a RalA/B activator.

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.

MORG1 limits mTORC1 signaling by inhibiting Rag GTPases.

Autophagy, an important quality control and recycling process vital for cellular homeostasis, is tightly regulated. The mTORC1 signaling pathway regulates autophagy under conditions of nutrient availability and scarcity. However, how mTORC1 activity is fine-tuned during nutrient availability to allow basal autophagy is unclear. Here, we report that the WD-domain repeat protein MORG1 facilitates basal constitutive autophagy by inhibiting mTORC1 signaling through Rag GTPases. Mechanistically, MORG1 interacts with active Rag GTPase complex inhibiting the Rag GTPase-mediated recruitment of mTORC1 to the lysosome. MORG1 depletion in HeLa cells increases mTORC1 activity and decreases autophagy. The autophagy receptor p62/SQSTM1 binds to MORG1, but MORG1 is not an autophagy substrate. However, p62/SQSTM1 binding to MORG1 upon re-addition of amino acids following amino acid's depletion precludes MORG1 from inhibiting the Rag GTPases, allowing mTORC1 activation. MORG1 depletion increases cell proliferation and migration. Low expression of MORG1 correlates with poor survival in several important cancers.

Characterization of Three Somatic Mutations in the 3'UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progressive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3' untranslated region (3'UTR) affecting positions +26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3'UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.

Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases.

Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

Raptor mediates the selective inhibitory effect of cardamonin on RRAGC-mutant B cell lymphoma.

BACKGROUND: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations. METHODS: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model. RESULTS: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment. CONCLUSIONS: Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.

Hepatitis B virus promotes its own replication by enhancing RAB5A-mediated dual activation of endosomal and autophagic vesicle pathways.

Chronic hepatitis B virus (HBV) infection remains one of the major global public health concerns, and it develop into liver fibrosis, cirrhosis, and hepatocellular carcinoma. Recent evidence suggests that endosomal and autophagic vesicles are beneficial for HBV replication. However, it has not been well elucidated how HBV exploits such intracellular vesicle systems for its replication. RAB5A, a member of small GTPase family, plays crucial roles in early endosome biogenesis and autophagy initiation. We observed that RAB5A mRNA and protein levels were significantly increased in HBV-expressing hepatoma cell lines as well as in liver tissue samples from chronic HBV-infected patients. Moreover, RAB5A silencing inhibited HBV replication and subviral particle (SVP) expression significantly in HBV-transfected and -infected hepatoma cells, whereas RAB5A overexpression increased them. Mechanistically, RAB5A increases HBV replication through enhancement of early endosome (EE) - late endosome (LE) activation by interacting with EEA1, as well as enhancing autophagy induction by interacting with VPS34. Additionally, HBV infection enhances RAB5A-mediated dual activation of EE-LE system and autophagy. Collectively, our findings highlight that HBV utilizes RAB5A-mediated dual activation of endosomal and autophagic vesicle pathways for its own replication and persistence. Therefore, RAB5A is a potential target for chronic HBV infection treatment.

Control of the flagellation pattern in Helicobacter pylori by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.