Pathophysiological mechanisms underlying the development of focal cortical dysplasia and their association with epilepsy: Experimental models as a research approach.

Focal cortical dysplasia (FCD) is a structural lesion that is the most common anatomical lesion identified in children, and the second most common in adults with drug-resistant focal-onset epilepsy. These lesions vary in size, location, and histopathological manifestations. FCDs are classified into three subtypes associated with loss-of-function mutations in PI3K/AKT, TSC1/TSC2, RHEB, and DEPDC/NPRL2/NPRL3. During the decades of research into FCD, experimental models have played an irreplaceable role in the research design of studies investigating disease pathogenesis, pathophysiology, and treatment. Further, the establishment of FCD experimental models has moved the field forward by (1) revealing the cellular processes and signaling pathways underlying FCD pathogenesis and (2) varying the methods and materials to study the function of FCD proteins. Currently, FCD experimental models are predominantly murine, with each model providing unique insights into FCD lesions. This review briefly summarizes the pathology and molecular functions of FCD, further comparing the available modeling methods and indexes, as well as the utilization of models, followed by an analysis of the similarities, advantages, and disadvantages between these models and human FCD.

NPRL2 is required for proliferation of oncogenic Ras-transformed bronchial epithelial cells.

Nitrogen permease regulator-like 2 (NPRL2/TUSC4) is known to exert both tumor-suppressing and oncogenic effects in different types of cancers, suggesting that its actions are context dependent. Here, we delineated the molecular and functional effects of NPRL2 in malignantly transformed bronchial epithelial cells. To do so, we depleted NPRL2 in oncogenic HRas-transduced and malignantly transformed human bronchial epithelial (BEAS2B), Ras-AI-T2 cells. Intriguingly, depletion of NPRL2 in these cells induced activation of mTORC1 downstream signaling, inhibited autophagy, and impaired Ras-AI-T2 cell proliferation both in vitro and in vivo. These results suggest that NPRL2 is required for oncogenic HRas-induced cell transformation. Depletion of NPRL2 increased levels of the DNA damage marker γH2AX, the cell cycle inhibitors p21 and p27, and the apoptosis marker cleaved-PARP. These NPRL2-depleted cells first accumulated at G1 and G2, and later exhibited signs of mitotic catastrophe, which implied that NPRL2 depletion may be detrimental to oncogenic HRas-transformed cells. Additionally, NPRL2 depletion reduced heat shock factor 1/heat shock element- and NRF2/antioxidant response element-directed luciferase reporter activities in Ras-AI-T2 cells, indicating that NPRL2 depletion led to the suppression of two key cytoprotective processes in oncogenic HRas-transformed cells. Overall, our data suggest that oncogenic HRas-transduced and malignantly transformed cells may depend on NPRL2 for survival and proliferation, and depletion of NPRL2 also induces a stressed state in these cells.

GATOR1 Mutations Impair PI3 Kinase-Dependent Growth Factor Signaling Regulation of mTORC1.

GATOR1 (GAP Activity TOward Rag 1) is an evolutionarily conserved GTPase-activating protein complex that controls the activity of mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid availability in cells. Genetic mutations in the GATOR1 subunits, NPRL2 (nitrogen permease regulator-like 2), NPRL3 (nitrogen permease regulator-like 3), and DEPDC5 (DEP domain containing 5), have been associated with epilepsy in humans; however, the specific effects of these mutations on GATOR1 function and mTORC1 regulation are not well understood. Herein, we report that epilepsy-linked mutations in the NPRL2 subunit of GATOR1, NPRL2-L105P, -T110S, and -D214H, increase basal mTORC1 signal transduction in cells. Notably, we show that NPRL2-L105P is a loss-of-function mutation that disrupts protein interactions with NPRL3 and DEPDC5, impairing GATOR1 complex assembly and resulting in high mTORC1 activity even under conditions of amino acid deprivation. Furthermore, our studies reveal that the GATOR1 complex is necessary for the rapid and robust inhibition of mTORC1 in response to growth factor withdrawal or pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). In the absence of the GATOR1 complex, cells are refractory to PI3K-dependent inhibition of mTORC1, permitting sustained translation and restricting the nuclear localization of TFEB, a transcription factor regulated by mTORC1. Collectively, our results show that epilepsy-linked mutations in NPRL2 can block GATOR1 complex assembly and restrict the appropriate regulation of mTORC1 by canonical PI3K-dependent growth factor signaling in the presence or absence of amino acids.

Clinical phenotype and genotype of NPRL2-related epilepsy: Four cases reports and literature review.

BACKGROUND: NPRL2-related epilepsy, caused by pathogenic germline variants of the NPRL2 gene, is a newly discovered childhood epilepsy linked to enhanced mTORC1 signalling. However, the phenotype and genotype of NPRL2 variants are still poorly understood. Here, we summarize the association between the phenotype and genotype of NPRL2-related epilepsy. METHODS: A retrospective analysis was conducted for four Chinese children with epilepsy due to likely pathogenic NPRL2 variants identified through whole-exome sequencing (WES). Previous reports of patients with NPRL2-related epilepsy were reviewed systematically. RESULTS: One of our patients presented focal epilepsy involving the central region, which should be distinguished from self-limited epilepsy with centrotemporal spikes (SeLECTS). The four novel likely pathogenic NPRL2 variants consisted of two nonsense variants, one frameshift variant, and one copy number variant (CNV). Bioinformatics analysis revealed the two nonsense variants to be highly conserved and cause alterations in protein structure. Including our four cases, a total of 33 patients with NPRL2-related epilepsy have been identified to date. The most common presentation is focal epilepsy (70%), including sleep-related hypermotor epilepsy (SHE), temporal lobe epilepsy (TLE), and frontal lobe epilepsy (FLE). Infantile epileptic spasms syndrome (IESS) is also a notable feature of NPRL2-related epilepsy. Malformations of cortical development (MCD, 8/20), especially focal cortical dysplasia (FCD, 6/20), are common neuroimaging abnormalities. Two-thirds of the NPRL2 variants reported are loss of function (LoF) (14/21). Among these mutations, c.100C>T (p.Arg34*) and c.314T>C (p.Leu105Pro) have been detected in two families (likely due to a founder effect). CONCLUSION: NPRL2-related epilepsy shows high phenotypic and genotypic heterogeneity. Our study expands the genotype spectrum of NPRL2-related epilepsy, and the phenotype of focal epilepsy involving the central region should be clearly distinguished with SeLECTS, with reference value for clinical diagnosis.

PRMT1 orchestrates with SAMTOR to govern mTORC1 methionine sensing via Arg-methylation of NPRL2.

Methionine is an essential branch of diverse nutrient inputs that dictate mTORC1 activation. In the absence of methionine, SAMTOR binds to GATOR1 and inhibits mTORC1 signaling. However, how mTORC1 is activated upon methionine stimulation remains largely elusive. Here, we report that PRMT1 senses methionine/SAM by utilizing SAM as a cofactor for an enzymatic activity-based regulation of mTORC1 signaling. Under methionine-sufficient conditions, elevated cytosolic SAM releases SAMTOR from GATOR1, which confers the association of PRMT1 with GATOR1. Subsequently, SAM-loaded PRMT1 methylates NPRL2, the catalytic subunit of GATOR1, thereby suppressing its GAP activity and leading to mTORC1 activation. Notably, genetic or pharmacological inhibition of PRMT1 impedes hepatic methionine sensing by mTORC1 and improves insulin sensitivity in aged mice, establishing the role of PRMT1-mediated methionine sensing at physiological levels. Thus, PRMT1 coordinates with SAMTOR to form the methionine-sensing apparatus of mTORC1 signaling.

[The regulatory relationship between RagA and Nprl2 in Drosophila gut development].

The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.

Risk signature identification and NPRL2 affects sunitinib sensitivity in clear cell renal cell carcinoma.

Tumor suppressor genes (TSGs) play a crucial role in tumorigenesis and drug resistance. We analyzed the subtypes of clear cell renal cell carcinoma (ccRCC) mediated by 8 genes contained in the 3p21.3 tumor suppressor gene cluster and their effects on TME cell infiltration based on the TCGA database. The risk score model was established by principal component analysis. The hub gene NPRL2 was selected by protein-protein interactions (PPI) analysis. The effect of NPRL2 on sunitinib sensitivity of ccRCC was verified by using CCK-8, colony formation assay, wound healing assay, transwell assay and xenograft tumor model. Changes in protein expression were detected by Western blotting. We found that 8 TSGs were all differentially expressed in ccRCC samples, which could divide ccRCC into two subtypes. The constructed risk score model could predict the prognosis and drug sensitivity of ccRCC patients, and was an independent prognostic factor for ccRCC. Over-expression of NPRL2 promoted apoptosis, inhibited EMT, decreased the phosphorylation of the PI3K/AKT/mTOR signaling pathway to inhibit its activity, and promoted the sensitivity of sunitinib to ccRCC cells. Collectively, our findings increased the understanding of TSGs in ccRCC, suggesting that NPRL2 as a TSG could enhance sunitinib sensitivity to ccRCC cells.

Novel GATOR1 variants in focal epilepsy.

INTRODUCTION: Variants in GATOR1 genes are well established in focal epilepsy syndromes. A strong association of GATOR1 variants with drug-resistant epilepsy as well as an increased risk of sudden unexplained death in epilepsy warrants developing strategies to facilitate the identification of patients who could potentially benefit from genetic testing and precision medicine. We aimed to determine the yield of GATOR1 gene sequencing in patients with focal epilepsy typically referred for genetic testing, establish novel GATOR1 variants and determine clinical, electroencephalographic, and radiological characteristics of variant carriers. PATIENTS AND METHODS: Ninety-six patients with clinical suspicion of genetic focal epilepsy with previous comprehensive diagnostic epilepsy evaluation in The Neurology Clinic, University Clinical Center of Serbia, were included in the study. Sequencing was performed using a custom gene panel encompassing DEPDC5, NPRL2, and NPRL3. Variants of interest (VOI) were classified according to criteria proposed by the American College of Medical Genetics and the Association for Molecular Pathology. RESULTS: Four previously unreported VOI in 4/96 (4.2%) patients were found in our cohort. Three likely pathogenic variants were determined in 3/96 (3.1%) patients, one frameshift variant in DEPDC5 in a patient with nonlesional frontal lobe epilepsy, one splicogenic DEPDC5 variant in a patient with nonlesional posterior quadrant epilepsy, and one frameshift variant in NPRL2 in a patient with temporal lobe epilepsy associated with hippocampal sclerosis. Only one VOI, a missense variant in NPRL3, found in 1/96 (1.1%) patients, was classified as a variant of unknown significance. CONCLUSION: GATOR1 gene sequencing was diagnostic in 3.1% of our cohort and revealed three novel likely pathogenic variants, including a previously unreported association of temporal lobe epilepsy with hippocampal sclerosis with an NPRL2 variant. Further research is essential for a better understanding of the clinical scope of GATOR1 gene-associated epilepsy.

Ring domains are essential for GATOR2-dependent mTORC1 activation.

The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.

Bioinformatics analysis of the prognostic and immunotherapeutic significance of NPRL2 in stomach adenocarcinoma.

Background: Stomach adenocarcinoma (STAD) is a major type of gastric cancer with high morbidity and mortality. NPRL2, a candidate cancer suppressor gene, has been shown to have anti-cancer effects in various types of cancers. Therefore, comprehensive analyses of NPRL2 in STAD may provide a potential prognostic marker and clinical target for the management of gastric cancer. Methods: Genomic expression and methylation were analysed based on data from the Human Protein Atlas, Gene Expression Omnibus and Oncomine database. Survival analyses were conducted with the Kaplan-Meier method, using data from The Cancer Genome Atlas database. Immune correlation analyses and prediction of response to immunotherapy were performed using the online Immune Cell Abundance Identifier. Co-expression analyses, functional clustering analyses and construction of a prognostic risk model were conducted in R, with the clinical covariates balanced by the inverse probability treatment weighting method. Results: NPRL2 was abnormally downregulated in STAD (P<0.05). Survival analysis highlighted a positive association between the expression of NPRL2 and clinical outcomes for patients (P<0.05). Based on co-expression analyses, we found that NPRL2 may be involved in epithelial-mesenchymal transition, gastric cancer stem cells, and responsiveness to chemotherapeutic agents in STAD (P<0.05). Furthermore, functional clustering analysis revealed that NPRL2 was involved in the mTOR signalling pathway, autophagy, and the amino acid starvation response (adjust P<0.05). In addition, NPRL2 was negatively associated with tumour-infiltrating immune cells while positively associated with immunotherapeutic biomarkers in STAD (P<0.05). Meanwhile, patients with high NPRL2 expression were predicted to have a better response to immunotherapy (P<0.05). Finally, a prognostic model constructed based on NPRL2-related genes could predict the prognosis of STAD patients (AUC =0.641), and the risk score was an independent prognostic factor for STAD patients (HR =4.855, 95% CI: 2.683-8.785, P<0.001). Conclusions: The present study provided a comprehensive analysis of the role and potential mechanisms of NPRL2 in STAD, suggesting that NPRL2 is a potential biomarker for the survival and prediction of immunotherapy response in STAD.

NPRL2 Inhibition of mTORC1 Controls Sodium Channel Expression and Brain Amino Acid Homeostasis.

Genetic mutations in nitrogen permease regulator-like 2 (NPRL2) are associated with a wide spectrum of familial focal epilepsies, autism, and sudden unexpected death of epileptics (SUDEP), but the mechanisms by which NPRL2 contributes to these effects are not well known. NPRL2 is a requisite subunit of the GAP activity toward Rags 1 (GATOR1) complex, which functions as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1) kinase when intracellular amino acids are low. Here, we show that loss of NPRL2 expression in mouse excitatory glutamatergic neurons causes seizures before death, consistent with SUDEP in humans with epilepsy. Additionally, the absence of NPRL2 expression increases mTORC1-dependent signal transduction and significantly alters amino acid homeostasis in the brain. Loss of NPRL2 reduces dendritic branching and increases the strength of electrically stimulated action potentials (APs) in neurons. The increased AP strength is consistent with elevated expression of epilepsy-linked, voltage-gated sodium channels in the NPRL2-deficient brain. Targeted deletion of NPRL2 in primary neurons increases the expression of sodium channel Scn1A, whereas treatment with the pharmacological mTORC1 inhibitor called rapamycin prevents Scn1A upregulation. These studies demonstrate a novel role of NPRL2 and mTORC1 signaling in the regulation of sodium channels, which can contribute to seizures and early lethality.

Phenotypic and Genotypic Characterization of NPRL2-Related Epilepsy: Two Case Reports and Literature Review.

The phenotype of nitrogen permease regulator-like 2 (NPRL2) gene-related epilepsy clinically manifests as a range of epilepsy syndromes, including familial focal epilepsy with variable foci (FFEVF), sleep-related hypermotor epilepsy (SHE), temporal lobe epilepsy (TLE), frontal lobe epilepsy (FLE), and infantile spasms (IS). The association between phenotype and genotype of NPRL2 variants has not been widely explored. This study aimed to explore the phenotype and genotype spectrum of NPRL2-related epilepsy. Here, we presented two clinical cases with NPRL2-related epilepsy, and discussed the characteristics, diagnosis, and treatment processes in the context of existing literature. Two novel NPRL2 likely pathogenic variants were identified by next-generation sequencing, including one splicing mutation (c.933-1G>A), and one frameshift mutation (c.257delG). The results of literature review showed that there were a total of 20 patients with NPRL2-related epilepsy whose mutations were mostly missense and hereditary. These findings indicate that the possibility of NPRL2 gene mutations in focal epilepsy should be considered for patients with family history, and that patients carrying different NPRL2 variants have different clinical manifestations. Our study expanded the genotype spectrum of NPRL2 and suggested that the type of NPRL2 variants might provide important information for the prognosis evaluation.

FOXO1 inhibits prostate cancer cell proliferation via suppressing E2F1 activated NPRL2 expression.

Previous studies in our lab suggest that nitrogen permease regulator 2-like (NPRL2) upregulation in prostate cancer is associated with malignant behavior and poor prognosis. However, the underlying mechanisms of NPRL2 dysregulation remain poorly understood. This study aimed to explore the transcription factors (TFs) contributing to NPRL2 dysregulation in prostate cancer. Potential TFs were identified using prostate tissue/cell-specific chromatin immunoprecipitation (ChIP)-seq data collected in the Cistrome Data Browser and Signaling Pathways Project. Dual-luciferase assay and ChIP-qPCR assay were conducted to assess the binding and activating effect of TFs on the gene promoter. Cell Counting Kit-8 and colony formation assays were performed to assess cell proliferation. Results showed that E2F1 is a TF that bound to the NPRL2 promoter and activated its transcription. NPRL2 inhibition significantly alleviated E2F1 enhanced cell proliferation. Kaplan-Meier survival analysis indicated that E2F1 upregulation was associated with unfavorable progression-free survival and disease-specific survival. FOXO1 interacted and E2F1 in both PC3 and LNCaP cells and weakened the binding of E2F1 to the NPRL2 promoter. Functionally, FOXO1 overexpression significantly slowed the proliferation of PC3 and LNCaP cells and also decreased E2F1 enhanced cell proliferation. In summary, this study revealed a novel FOXO1/E2F1-NPRL2 regulatory axis in prostate cancer. E2F1 binds to the NPRL2 promoter and activates its transcription, while FOXO1 interacts with E2F1 and weakens its transcriptional activating effects. These findings help expand our understanding of the prostate cancer etiology and suggest that the FOXO1/E2F1-NPRL2 signaling axis might be a potential target.

NPRL2 reduces the niraparib sensitivity of castration-resistant prostate cancer via interacting with UBE2M and enhancing neddylation.

In this study, we explored the regulatory effects of nitrogen permease regulator 2-like (NPRL2) on niraparib sensitivity, a PARP inhibitor (PARPi) in castrate-resistant prostate cancer (CRPC). Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program were retrospectively examined. Gene-set enrichment analysis (GSEA) was conducted between high and low NRPL2 expression prostate adenocarcinoma (PRAD) cases in TCGA. CCK-8 assay, Western blot analysis of apoptotic proteins, and flow cytometric analysis of apoptosis were applied to test niraparib sensitivity. Immunofluorescent (IF) staining and co-immunoprecipitation (co-IP) were conducted to explore the proteins interacting with NPRL2. Results showed that the upregulation of a canonical protein-coding transcript of NPRL2 (ENST00000232501.7) is associated with an unfavorable prognosis. Bioinformatic analysis predicts a physical interaction between NPRL2 and UBE2M, which is validated by a following Co-IP assay. This interaction increases NPRL2 stability by reducing polyubiquitination and proteasomal degradation. Depletion of NPRL2 or UBE2M significantly increases the niraparib sensitivity of CRPC cells and enhances niraparib-induced tumor growth inhibition in vivo. NPRL2 cooperatively enhances UBE2M-mediated neddylation and facilitates the degradation of multiple substrates of Cullin-RING E3 ubiquitin ligases (CRLs). In conclusion, this study identified a novel NPRL2-UBE2M complex in modulating neddylation and niraparib sensitivity of CRPC cells. Therefore, targeting NPRL2 might be considered as an adjuvant strategy for PARPi therapy.

NPRL2 promotes docetaxel chemoresistance in castration resistant prostate cancer cells by regulating autophagy through the mTOR pathway.

Docetaxel-based chemotherapy is recommended for metastatic castration-resistant prostate cancer (mCRPC). However, chemoresistance is inevitable and eventually progresses after several rounds of chemotherapy. Therefore, exploration of new therapeutic targets and molecular mechanisms that contribute to chemoresistance remains necessary. Our previous study accidentally demonstrated that expression of nitrogen permease regulator-like 2 (NPRL2), which is defined as a tumor suppressor, is upregulated in prostate cancer (PCa) and linked to poor prognosis, particularly in CRPC. The aim of this study was to investigate the role of NPRL2 in the chemoresistant CRPC cells. We found that NPRL2 was significantly overexpressed in docetaxel-resistant CRPC cells, while autophagy was enhanced and mTOR signaling was inhibited. Inhibiting NPRL2 increased the sensitivity to docetaxel in docetaxel-resistant CRPC cells, enhanced apoptosis and inhibited autophagy, and the opposite trends were observed when the mTOR inhibitor torin 1 was added to NPRL2-silenced cells. We further found that NPRL2 silenced docetaxel-resistant CRPC cells were sensitive to docetaxel in vivo. Briefly, our research reveals that overexpression of NPRL2 promotes chemoresistance by regulating autophagy via mTOR signaling and inhibits apoptosis in CRPC cells.

Nitrogen Permease Regulator-Like-2 Exhibited Anti-Tumor Effects And Enhanced The Sensitivity Of Colorectal Cancer Cells To Oxaliplatin And 5-Fluorouracil.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Our previous study revealed that nitrogen permease regulator-like-2 (NPRL2), a promising anti-tumor gene, was downregulated at both the blood and tissue levels in CRC patients compared with that in healthy individuals. PURPOSE: This study aims to explore the role of NPRL2 in CRC. METHODS: Herein, we constructed NPRL2 overexpression lentivirus vectors and transfected them into HT29 cells. The transfected cells were inoculated subcutaneously into nude mice. Tumor growth, pathology, apoptosis, and the protein expression of caspase-3, caspase-7, Bax, Bcl-2, and phosphorylated protein kinase B (p-Akt) were evaluated. To further explore whether NPRL2 could reduce drug resistance of CRC cells against oxaliplatin (L-OHP) and 5-fluorouracil (5-FU), we constructed a tumor model using HT29 cells. The tumor model was treated with lentiviral particles assembled with vectors encoding NPRL2 and exposed to L-OHP and 5-FU. Tumor growth, pathology, apoptosis, and the protein expression of caspase-3, caspase-7, Bax, Bcl-2, p-Akt, P-glycoprotein (P-gp), and multidrug resistance protein 1 (MRP1) were evaluated. RESULTS: The results indicated that in the in vivo CRC xenograft model, NPRL2 reduced the tumor volume and weight and enhanced apoptosis. Our results also confirmed that NPRL2 enhanced the sensitivity of CRC cells to L-OHP and 5-FU. Our studies further demonstrated that NPRL2 exerted anti-tumor and anti-drug resistance effects through the caspase-3, caspase-7, Bax, Bcl-2, Akt, P-gp, and MRP1 pathways. CONCLUSION: Our present work demonstrated that NPRL2 exhibited anti-tumor effects and enhanced the sensitivities of CRC cells to L-OHP and 5-FU through the P-gp and MRP1 pathways.

The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila.

Aging and age-related diseases occur in almost all organisms. Recently, it was discovered that the inhibition of target of rapamycin complex 1 (TORC1), a conserved complex that mediates nutrient status and cell metabolism, can extend an individual's lifespan and inhibit age-related diseases in many model organisms. However, the mechanism whereby TORC1 affects aging remains elusive. Here, we use a loss-of-function mutation in nprl2, a component of GATOR1 that mediates amino acid levels and inhibits TORC1 activity, to investigate the effect of increased TORC1 activity on the occurrence of age-related digestive dysfunction in Drosophila. We found that the nprl2 mutation decreased Drosophila lifespan. Furthermore, the nprl2 mutant had a distended crop, with food accumulation at an early age. Interestingly, the inappropriate food distribution and digestion along with decreased crop contraction in nprl2 mutant can be rescued by decreasing TORC1 activity. In addition, nprl2-mutant flies exhibited age-related phenotypes in the midgut, including short gut length, a high rate of intestinal stem cell proliferation, and metabolic dysfunction, which could be rescued by inhibiting TORC1 activity. Our findings showed that the gastrointestinal tract aging process is accelerated in nprl2-mutant flies, owing to high TORC1 activity, which suggested that TORC1 promotes digestive tract senescence.

TOR Signaling in Caenorhabditis elegans Development, Metabolism, and Aging.

The Target of Rapamycin (TOR or mTOR) is a serine/threonine kinase that regulates growth, development, and behaviors by modulating protein synthesis, autophagy, and multiple other cellular processes in response to changes in nutrients and other cues. Over recent years, TOR has been studied intensively in mammalian cell culture and genetic systems because of its importance in growth, metabolism, cancer, and aging. Through its advantages for unbiased, and high-throughput, genetic and in vivo studies, Caenorhabditis elegans has made major contributions to our understanding of TOR biology. Genetic analyses in the worm have revealed unexpected aspects of TOR functions and regulation, and have the potential to further expand our understanding of how growth and metabolic regulation influence development. In the aging field, C. elegans has played a leading role in revealing the promise of TOR inhibition as a strategy for extending life span, and identifying mechanisms that function upstream and downstream of TOR to influence aging. Here, we review the state of the TOR field in C. elegans, and focus on what we have learned about its functions in development, metabolism, and aging. We discuss knowledge gaps, including the potential pitfalls in translating findings back and forth across organisms, but also describe how TOR is important for C. elegans biology, and how C. elegans work has developed paradigms of great importance for the broader TOR field.

Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the Rag GTPases.

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex.

NPRL2 enhances autophagy and the resistance to Everolimus in castration-resistant prostate cancer.

BACKGROUND: Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and involved in the mTOR signaling and drug resistance in several cancers. However, the role of NPRL2 in regulating the resistance to Everolimus (EVS), an inhibitor of the mTOR, in castration-resistant prostate cancer (CRPC) is still unclear. Therefore, in present study, we evaluated the role of NPRL2 and its potential resistance to EVS in CRPC. METHODS: NPRL2 expression levels in prostate tissues, including benign prostate hyperplasia (BPH) tissues, primary prostate cancer (PCa) tissues, CRPC tissues, and several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) were be evaluated by immunohistochemistry, RT-PCR, and Western blot. Furthermore, we employed the loss or gain function of NPRL2 to determine the role of NPRL2 in regulating the proliferation, sensitivity to EVS, the mTOR signaling, autophagy in CRPC. Lastly, relationship between NPRL2 expression level and the efficacy of EVS were evaluated in mice tumor xenograft models. RESULTS: NPRL2 expression level is upregulated in PCa, particularly in the CRPC. NPRL2 over-expression promoted the proliferation, resistance to EVS, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to EVS in PC3 and LNPER cells. Moreover, NPRL2-silencing increased the activity of mTOR signaling, and the autophagy attenuation induced by NPRL2-silencing in EVS-treated CRPC cells was associated with the increase of apoptosis. In addition, the growth prevention of NPRL2-silencing LNPER tumors in mice induced by EVS-treatment was associated with the autophagy attenuation and apoptosis increase. CONCLUSIONS: NPRL2 may act as a pro-growth factor in PCa. The high levels of NPRL2 expression in CRPC promote resistance to EVS by enhancing autophagy. NPRL2 may be a new therapeutic target for intervention of CRPC and a biomarker for predicting resistance to EVS in CRPC.