K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells.

Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell's behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5 integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed traffic remain poorly annotated. Here we identify an important role for the GTPase-activating protein p120RasGAP in ECs, promoting the recycling of α5 integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5 integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, resulted in the accumulation of α5 integrin in early endosomes, a loss of α5 integrin from surface adhesions, and attenuated EC polarisation. Endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors co-traffic internalised cargoes. Importantly, we utilise comparative proteomics analyses to isolate a comprehensive map of NRP1-dependent and NRP2-dependent α5 integrin interactions in ECs.

SHP2-EGFR States in Dephosphorylation Can Inform Selective SHP2 Inhibitors, Dampening RasGAP Action.

SHP2 is a positive regulator of the EGFR-dependent Ras/MAPK pathway. It dephosphorylates a regulatory phosphorylation site in EGFR that serves as the binding site to RasGAP (RASA1 or p120RasGAP). RASA1 is activated by binding to the EGFR phosphate group. Active RASA1 deactivates Ras by hydrolyzing Ras-bound GTP to GDP. Thus, SHP2 dephosphorylation of EGFR effectively prevents RASA1-mediated deactivation of Ras, thereby stimulating proliferation. Despite knowledge of this vital regulation in cell life, mechanistic in-depth structural understanding of the involvement of SHP2, EGFR, and RASA1 in the Ras/MAPK pathway has largely remained elusive. Here we elucidate the interactions, the factors influencing EGFR's recruitment of RASA1, and SHP2's recognition of the substrate site in EGFR. We reveal that RASA1 specifically interacts with the DEpY992LIP motif in EGFR featuring a proline residue at the +3 position C-terminal to pY primarily through its nSH2 domain. This interaction is strengthened by the robust attraction of two acidic residues, E991 and D990, of EGFR to two basic residues in the BC-loop near the pY-binding pocket of RASA1's nSH2. In the stable precatalytic state of SHP2 with EGFR (DADEpY992LIPQ), the E-loop of SHP2's active site favors the interaction with the (-2)-position D990 and (-4)-position D988 N-terminal to pY992 in EGFR, while the pY-loop constrains the (+4)-position Q996 C-terminal to pY992. These specific interactions not only provide a structural basis for identifying negative regulatory sites in other RTKs but can inform selective, high-affinity active-site SHP2 inhibitors tailored for SHP2 mutants.

A temporally-restricted pattern of endothelial cell collagen 4 alpha 1 expression during embryonic development determined with a novel knockin Col4a1-P2A-eGFP mouse line.

Classical collagen type IV comprising of a heterotrimer of two collagen IV alpha 1 chains and one collagen IV alpha 2 chain is the principal type of collagen synthesized by endothelial cells (EC) and is a major constituent of vascular basement membranes. In mouse and man, mutations in genes that encode collagen IV alpha 1 and alpha 2 result in vascular dysfunction. In addition, mutations in genes that encode the Ephrin receptor B4 (EPHB4) and the p120 Ras GTPase-activating protein (RASA1) that cause increased activation of the Ras mitogen-activated protein kinase (MAPK) signaling pathway in EC result in vascular dysfunction as a consequence of impaired export of collagen IV. To understand the pathogenesis of collagen IV-related vascular diseases and phenotypes it is necessary to identify at which times collagen IV is actively synthesized by EC. For this purpose, we used CRISPR/Cas9 targeting in mice to include immediately after the terminal Col4a1 codon a sequence that specifies a P2A peptide followed by enhanced green fluorescent protein (eGFP). Analysis of eGFP expression in Col4a1-P2A-eGFP mice revealed active embryonic EC synthesis of collagen IV alpha 1 through mid to late gestation followed by a sharp decline before birth. These results provide a contextual framework for understanding the basis for the varied vascular abnormalities resulting from perturbation of EC expression and export of functional collagen IV.

Clear Cell Squamous Cell Carcinoma of the Maxillary Gingiva Associated with PIK3CA and HRAS Mutations: Report of a Case and Literature Review.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common oral malignancy, and somatic mutations in some driver genes have been implicated in SCC development. Clear cell SCC (CCSCC) is a rare histological variant of SCC, and various clear cell neoplasms must be considered in the differential diagnosis of CCSCC in the oral cavity. Based on a limited number of CCSCC cases reported in the oral cavity, CCSCC is considered an aggressive variant of SCC with a poor prognosis; however, its genetic characteristics remain unknown. METHODS: A maxillary gingival tumor in an 89-year-old female was described and investigated using immunohistochemical staining, special staining, fluorescence in situ hybridization, and next-generation sequencing (NGS) with a custom panel of driver genes, including those associated with SCC and clear cell neoplasm development. RESULTS: Histopathological examination revealed a proliferation of atypical epithelial cells with abundant clear cytoplasm and enlarged and centrally placed round nuclei. The tumor was exophytic with deep, penetrating proliferation. The atypical clear cells were continuous with the conventional SCC cells. Immunohistochemical analysis showed that the clear cells were positive for CK AE1/AE3 and CK5/6 and nuclear-positive for p63. In contrast, the clear cells were negative for αSMA, S100, HMB45, Melan-A, CD10, and p16. p53 immunoreactivity exhibited a wild-type expression pattern. Additionally, the clear cells were positive for periodic acid-Schiff (PAS) and negative for diastase-PAS, mucicarmine, and Alcian blue. Based on these results, the diagnosis of CCSCC was confirmed. Molecular analysis of the clear cells identified PIK3CA p.E542K (c.1624G>A) and HRAS p.G12A (c.35 G>C) somatic mutations classified as oncogenic. No pathogenic variants were identified in TP53, EWSR1, AKT1, PTEN, BRAF, KRAS, NRAS, RASA1, or MAML2. CONCLUSIONS: We report a case of CCSCC of the oral cavity with PIK3CA and HRAS mutations. The identification of PIK3CA and/or HRAS mutations is rare in SCC; however, both mutations are important potential targets for antitumor therapy. A detailed analysis of gene mutations in CCSCC may lead to a better understanding of its biological behavior and an improved prognosis, as well as a differential diagnosis from other clear cell neoplasms.

Regulation of IncRNA ZNF667-AS1 in Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells via Mediating ceRNA Network.

Esophageal squamous cell carcinoma (ESCC), classified as a primary histological subtype of esophageal cancer (EC), dominates approximately 90% of the newly diagnosed EC. Long non-coding RNAs (lncRNAs) are frequently related to the course of ESCC. The current study aimed to investigate whether lncRNA zinc finger protein 667-antisense RNA 1 (ZNF667-AS1) modulates the proliferation and invasion of ESCC cells. ESCC tissues and cell lines, para-carcinoma tissues, and human esophageal epithelial cells (HEEpiCs) were collected. lncRNA ZNF667-AS1 expression in the above tissues and cells was detected. The effect of lncRNA ZNF667-AS1 on proliferation and invasion of Eca109 cells was detected using cell counting kit-8, colony formation, and Transwell assays. lncRNA ZNF667-AS1 subcellular localization was determined via the nuclear/cytosol fractionation assay. The binding relationships between miR-18b-5p and lncRNA ZNF667-AS1 and RAS p21 protein activator 1 (RASA1) were verified using dual-luciferase reporter gene experiment and RNA immunoprecipitation experiment. The expressions of miR-18b-5p and RASA1 in the tissues and cells were identified. The roles of miR-18b-5p overexpression or silencing RASA1 in proliferation and invasion of ESCC cells were examined through rescue experiments. lncRNA ZNF667-AS1 was underexpressed in ESCC tissues and cells, and lncRNA ZNF667-AS1 overexpression hampered ESCC cell proliferation and invasiveness. miR-18b-5p targeted RASA1 while lncRNA ZNF667-AS1 promoted RASA1 transcription via binding to miR-18b-5p. Over-expression miR-18b-5p or silencing RASA1 reversed the inhibitory effects of lncRNA ZNF667-AS1 overexpression on ESCC cell proliferation and invasion. lncRNA ZNF667-AS1 overexpression accelerated RASA1 transcription by competitively binding to miR-18b-5p, thus suppressing ESCC cell proliferation and invasion.

SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP.

RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.

MicroRNA-4487 regulates vascular smooth muscle cell proliferation, migration and apoptosis by targeting RAS p21 protein activator 1.

BACKGROUND: Dysregulation of microRNA (miRNA) is involved in the pathogenesis of a variety of diseases, including atherosclerosis (AS). However, the role of miRNA-4487 (miR-4487) in the development of AS is not fully clarified. This study is intended to investigate the regulatory effects of miR-4487 on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) and the related mechanisms. METHODS: Oxidized low-density lipoprotein (ox-LDL) was employed to induce the dysfunction of VSMCs. Subsequently, miR-4487 expression was detected by quantitative real-time PCR. Afterward, the expression levels of RAS p21 protein activator 1 (RASA1) and apoptosis-related proteins (Bcl-2, Bax, Cleaved caspase 3, Cleaved caspase 9) were detected by Western blotting. The proliferation, migration and apoptosis of VSMCs were then detected by CCK-8, BrdU, Transwell and flow cytometry assays, respectively. Moreover, a dual-luciferase reporter gene assay was executed to verify the targeting between miR-4487 to the RASA1 3'-untranslated region (3'-UTR). RESULTS: ox-LDL treatment increased miR-4487 expression and decreased RASA1 expression in VSMCs. Additionally, ox-LDL treatment promoted the proliferation and migration of VSMCs, but inhibited apoptosis. Besides, the effects of ox-LDL treatment on the proliferation, migration and apoptosis of VSMCs were attenuated by the transfection of miR-4487 inhibitors. Furthermore, miR-4487 directly targeted the 3'-UTR of RASA1 mRNA and repressed the expression level of RASA1. Also, RASA1 knockdown reversed the effects of miR-4487 inhibition on VSMCs. CONCLUSION: MiR-4487 promotes VSMCs viability and migration and inhibits apoptosis by targeting RASA1 in VSMCs, by which it promotes the pathogenesis of AS.

Down-regulated placental miR-21 contributes to preeclampsia through targeting RASA1.

Human placenta was obtained from early onset preeclampsia, late onset preeclampsia, and their gestational age-matched normal pregnancy. Using RT-qPCR, western blot, and immunohistochemistry, it was demonstrated that miR-21 expressions were significantly decreased in preeclampsia while RASA1 were increased. Suppression of miR-21 in placental HTR-8/SVneo cells, remarkably upregulated RASA1, decreased proliferation, inhibited invasion, and promoted apoptosis of trophoblast cells, while overexpression of miR-21 alleviated these effects. Dual-luciferase reporter assays revealed RASA1 to be a direct target of miR-21 in trophoblast cells. miR-21 may serve key roles in the development of preeclampsia by targeting RASA1.

The long noncoding RNA MEG3 regulates Ras-MAPK pathway through RASA1 in trophoblast and is associated with unexplained recurrent spontaneous abortion.

BACKGROUND: Maternally Expressed Gene 3 (MEG3) is expressed at low levels in placental villi during preeclampsia; however, its roles in unexplained recurrent spontaneous abortion (URSA) remain unclear. In this study, we aimed to explore the relationship between MEG3 and URSA. METHODS: The differentially expressed lncRNAs (MEG3) and its downstream genes (RASA1) were identified using bioinformatics analysis of Genomic Spatial Event (GSE) database. The expression levels of MEG3 in embryonic villis (with gestational ages of 49-63 days) and primary trophoblasts were determined using quantitative RT-PCR assay. A mouse model of Embryo implantation, Cell Counting Kit-8 (CCK-8), flow cytometry, and Transwell migration assays were performed to determine the implantation, proliferative, apoptotic, and invasive capacities of trophoblast. The level of phosphorylated core proteins in the RAS-MAPK pathway were analyzed using Western blot assay. The mechanisms of MEG3 in the regulation of RASA1 were studied by RNA pulldown, RNA immunoprecipitation (RIP), DNA pulldown, and chromatin immunoprecipitation (ChIP) assays. RESULTS: MEG3 had a low expression level in embryonic villis of 102 URSA patients compared with those of 102 normal pregnant women. MEG3 could promote proliferation and invasion, inhibit the apoptosis of primary trophoblast of URSA patients (PT-U cells), as well as promote embryo implantation of mouse. Besides, MEG3 also promoted the phosphorylation of rapidly accelerated fibrosarcoma (Raf), mitogen-activated protein kinase kinase (MEK), and extracellular-signal-regulated kinase (ERK) proteins. The results of RNA pull down and RIP assays showed that MEG3 bound with the enhancer of zeste homolog 2 (EZH2). The DNA pulldown assay revealed that MEG3 could bind to the promoter sequence of the RAS P21 Protein Activator 1 (RASA1) gene. Further, the ChIP assay showed that MEG3 promoted the binding of EZH2 to the promoter region of the RASA1 gene. CONCLUSIONS: The inactivation of MEG3 in embryonic villi association with URSA; MEG3 inhibited the expression of RASA1 by mediating the histone methylation of the promoter of RASA1 gene by EZH2, thereby activating the RAS-MAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts.

Abnormal H3K27 histone methylation of RASA1 gene leads to unexplained recurrent spontaneous abortion by regulating Ras-MAPK pathway in trophoblast cells.

Some studies suggest that the inactivation of the Ras-MAPK pathway in trophoblast cells can lead to recurrent abortion, but the molecular mechanism underlying the inactivation of this pathway in trophoblast cells is still unclear. This study aimed to explore the relationship between the mechanism of abnormal activation of RASA1, a regulatory protein of the Ras-MAPK pathway, and unexplained recurrent spontaneous abortion. RT-qPCR was used to detect the transcription levels of RASA1 gene. Immunohistochemistry and Western blot were used to detect the expression levels of the RASA1, Raf and MEK proteins. CCK-8, TUNEL and Transwell assays were used to detect the proliferative, apoptotic, and invasive capacities of HTR-8/SVneo cells. ChIP assays were used to detect the enrichment of H3K27me3 in RASA1 gene promoter. Abortion villi experiments showed that the enrichment of H3K27me3 in the RASA1 gene promoter was reduced, and that both RASA1 gene transcription and RASA1 protein expression were increased. Cell experiments confirmed that RASA1 could decrease the phosphorylated Raf and MEK proteins, inhibit the proliferation and invasion ability, and promote the apoptosis ability of HTR-8/SVneo cells. It was also found that the proliferation and invasion ability as well as the Ras-MAPK pathway activity of HTR-8/SVneo cells were inhibited when treated with histone methyltransferase inhibitor DZNep. RASA1 gene was abnormally activated in unexplained recurrent spontaneous abortion villi due to the decreased enrichment of H3K27me3 in the gene promoter. High expression of RASA1 could inhibit the activity of the Ras-MAPK pathway, and thus inhibit the proliferation and invasion ability of trophoblast cells.

Inhibition of lncRNA SNHG8 plays a protective role in hypoxia-ischemia-reoxygenation-induced myocardial injury by regulating miR-335 and RASA1 expression.

Long non­coding (lnc)RNAs serve a role in a number of diseases, including different types of cancer and acute myocardial infarction. The aim of the present study was to investigate the protective role of lncRNA small nucleolar RNA host gene 8 (SNHG8) in hypoxia­ischemia­reoxygenation (HI/R)­induced myocardial injury and its potential mechanism of action. Cell viability, proliferation, creatine kinase myocardial band, cell apoptosis and protein expression levels were determined by Cell Counting Kit­8 assay, EdU assay, ELISA, flow cytometry and western blotting, respectively. The association between SNHG8 and microRNA (miR)­335 was confirmed using a dual­luciferase reporter gene assay. The effects of the miR­335 inhibitor transfections had on increasing apoptosis and decreasing H9C2 cell viability were reversed in cells co­transfected with SNHG8 small interfering (si)RNA. Furthermore, it was found that miR­335 could regulate RAS p21 protein activator 1 (RASA1) expression and that transfection with SNHG8 siRNA downregulated RASA1 expression. Silencing of RASA1 protected against HI/R­induced H9C2 cell injury. However, SNHG8 siRNA did not further reduce apoptosis, demonstrating that SNHG8 may act through RASA1, and RASA1 may mediate the protection of SNHG8 siRNA in HI/R myocardial injury. Thus, inhibition of lncRNA SNHG8 alleviated HI/R­induced myocardial damage by regulating miR­335 and RASA1.

The Role of Low Complexity Regions in Protein Interaction Modes: An Illustration in Huntingtin.

Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein-protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein-protein interactions with a large hub such as HTT when enough protein interaction data is available.

miR-31-5p promotes proliferation and inhibits apoptosis of goat hair follicle stem cells by targeting RASA1/MAP3K1 pathway.

The Yangtze River Delta white goat is a sole goat species that can naturally produce superior-quality brush hair. It's worth to mention that study the developmental mechanism of goat hair follicle stem cells is vital for future breed preservation and molecular breeding. In this study, we successfully isolated hair follicle stem cells from the skin tissue of fetal sheep neck spine, and harvested superior-quality and normal-quality brush hair goat tissue. The expression of miR-31-5p in goat hair follicle stem cells was verified by qPCR and Western blot. The effects of overexpression or inhibition of miR-31-5p on the proliferation and apoptosis of hair follicle stem cells were detected by EdU, CCK-8, flow cytometry, etc. miR-31-5p can significantly improve cell proliferation and inhibit cell apoptosis by targeting RASA1 and upregulating MAP3K1 level, whereas miR-31-5p knockdown led to an opposite effect. These results reveal a miR-31-5p-associated regulatory network between miR-31-5p and RASA1/MAP3K1 during the progression of superiorquality brush hair traits.

Role of RASA1 in cancer: A review and update (Review).

Ras p21 protein activator 1 (RASA1) is a regulator of Ras GDP and GTP and is involved in numerous physiological processes such as angiogenesis, cell proliferation, and apoptosis. As a result, RASA1 also contributes to pathological processes in vascular diseases and tumour formation. This review focuses on the role of RASA1 in multiple tumours types in the lung, intestines, liver, and breast. Furthermore, we discuss the potential mechanisms of RASA1 and its downstream effects through Ras/RAF/MEK/ERK or Ras/PI3K/AKT signalling. Moreover, miRNAs are capable of regulating RASA1 and could be a novel targeted treatment strategy for tumours.

Parkes Weber syndrome associated with two somatic pathogenic variants in RASA1.

Parkes Weber syndrome is associated with autosomal dominant inheritance, caused by germline heterozygous inactivating changes in the RASA1 gene, characterized by multiple micro arteriovenous fistulas and segmental overgrowth of soft tissue and skeletal components. The focal nature and variable expressivity associated with this disease has led to the hypothesis that somatic "second hit" inactivating changes in RASA1 are necessary for disease development. We report a 2-yr-old male with extensive capillary malformation and segmental overgrowth of his lower left extremity. Ultrasound showed subcutaneous phlebectasia draining the capillary malformation; magnetic resonance imaging showed overgrowth of the extremity with prominence of fatty tissues, fatty infiltration, and enlargement of all the major muscle groups. Germline RASA1 testing was normal. Later somatic testing from affected tissue showed two pathogenic variants in RASA1 consistent with the c.934_938del, p.(Glu312Argfs*14) and the c.2925del, p.(Asn976Metfs*20) with variant allele fractions of 3.6% and 4.2%, respectively. The intrafamilial variability of Parkes Weber syndrome involving segmental overgrowth of soft tissue, endothelium, and bone is strongly suggestive of a somatic second-hit model. There are at least two reports of confirmed second somatic hits in RASA1 To our knowledge, this is the first report of an individual with two somatic pathogenic variants in the RASA1 gene in DNA from a vascular lesion.

RASA1 inhibits the progression of renal cell carcinoma by decreasing the expression of miR-223-3p and promoting the expression of FBXW7.

RAS p21 protein activator 1 (RASA1), also known as p120-RasGAP, is a RasGAP protein that functions as a signaling scaffold protein, regulating pivotal signal cascades. However, its biological mechanism in renal cell carcinoma (RCC) remains unknown. In the present study, RASA1, F-box/WD repeat-containing protein 7 (FBXW7), and miR-223-3p expression were assessed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Then, the targeted correlations of miR-223-3p with FBXW7 and RASA1 were verified via a dual-luciferase reporter gene assay. CCK-8, flow cytometry, and Transwell assays were implemented independently to explore the impact of RASA1 on cell proliferation, apoptosis, migration, and cell cycle progression. Finally, the influence of RASA1 on tumor formation in RCC was assessed in vivo through the analysis of tumor growth in nude mice. Results showed that FBXW7 and RASA1 expression were decreased in RCC tissues and cell lines, while miR-223-3p was expressed at a higher level. Additionally, FBXW7 and RASA1 inhibited cell proliferation but facilitated the population of RCC cells in the G0/G1 phase. Altogether, RASA1 may play a key role in the progression of RCC by decreasing miR-223-3p and subsequently increasing FBXW7 expression.

The GTPase-activating protein p120RasGAP has an evolutionarily conserved "FLVR-unique" SH2 domain.

The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved FLVR motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket that appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position ßD4) and K400A (SH2 position ßD6) is required to disrupt the binding. These results indicate a hitherto unrecognized diversity in SH2 domain interactions with phosphotyrosine and classify the C-terminal SH2 domain of p120RasGAP as "FLVR-unique."

MicroRNA-144 relieves chronic constriction injury-induced neuropathic pain via targeting RASA1.

MicroRNAs (miRNAs) have been shown to participate in development of neuropathic pain. However, the role of microRNA-144 (miR-144) in neuropathic pain remains unclear. In the present study, we established a neuropathic pain mouse model via chronic constriction injury (CCI)-induction. The successful establishment of this model was confirmed via evaluation of paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). By using this model, we found that miR-144 was significantly downregulated in CCI-induced neuropathic pain mice. In addition, intrathecal injection of miR-144 agomiR alleviated mechanical and thermal hyperalgesia in neuropathic pain mice as shown by the increased of PWT and PWL. Moreover, miR-144 negatively regulated neuroinflammation by decreasing the expression of proinflammatory mediators, including TNF-α (tumor necrosis factor-α), IL (interleukin)-1ß, and IL-6, thus facilitating the inhibition of neuropathic pain development. Mechanistically, RASA1 (RAS P21 Protein Activator 1) was downregulated following the injection of agomiR-144, and was verified to be a target of miR-144. Furthermore, overexpression of RASA1 reversed the inhibitory effect of miR-144 on neuropathic pain. Therefore, the present study suggested that miR-144 has the potential to be explored as therapeutic target for treatment of neuropathic pain.

Alternative NF-κB signaling controls peripheral homeostasis and function of regulatory T cells.

Regulatory T cells (Tregs) maintain immune homeostasis and play an important role in tissue regeneration after injury. Mutations affecting development or homeostasis of Tregs lead to immune pathologies in humans and are often fatal in mouse models. Although the pathways required for Treg development are being increasingly characterized, factors crucial for Treg homeostasis are not completely understood. Previously we have found a role for alternative NF-κB pathway in restricting T cell activation and Th17 differentiation. Here, by using the mouse model of uncontrolled alternative NF-κB signaling we identify a crucial intrinsic role of RelB signaling in regulating homeostasis and competitive fitness of Tregs. The failure of p100-/- Tregs to maintain the population of effector Tregs and efficiently suppress immune reactions results in lethal multiorgan Th1-mediated inflammation in Rag1-/- recipients. This inflammation is combined with severe lymphopenia and could be rescued by adoptive transfer of wild type Tregs. Thus in addition to its role in Th17 differentiation, RelB acts as a potent inhibitor of Treg effector functions. Our results point to RelB as a potential therapeutic target for Treg manipulation.