Journal articles on the topic 'RagC'
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1
Gollwitzer, Peter, Nina Grützmacher, Sabine Wilhelm, Daniel Kümmel, and Constantinos Demetriades. "A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids." Nature Cell Biology 24, no. 9 (September 2022): 1394–406. http://dx.doi.org/10.1038/s41556-022-00976-y.
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AbstractAmino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.
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Lee, Minji, Jong Hyun Kim, Ina Yoon, Chulho Lee, Mohammad Fallahi Sichani, Jong Soon Kang, Jeonghyun Kang, et al. "Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): E5279—E5288. http://dx.doi.org/10.1073/pnas.1801287115.
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A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.
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Figlia, Gianluca, Sandra Müller, Anna M. Hagenston, Susanne Kleber, Mykola Roiuk, Jan-Philipp Quast, Nora ten Bosch, et al. "Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition." Nature Cell Biology 24, no. 9 (September 2022): 1407–21. http://dx.doi.org/10.1038/s41556-022-00977-x.
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AbstractMechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.
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Meng, Jin, and Shawn M. Ferguson. "GATOR1-dependent recruitment of FLCN–FNIP to lysosomes coordinates Rag GTPase heterodimer nucleotide status in response to amino acids." Journal of Cell Biology 217, no. 8 (May 30, 2018): 2765–76. http://dx.doi.org/10.1083/jcb.201712177.
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Folliculin (FLCN) is a tumor suppressor that coordinates cellular responses to changes in amino acid availability via regulation of the Rag guanosine triphosphatases. FLCN is recruited to lysosomes during amino acid starvation, where it interacts with RagA/B as a heterodimeric complex with FLCN-interacting proteins (FNIPs). The FLCN–FNIP heterodimer also has GTPase-activating protein (GAP) activity toward RagC/D. These properties raised two important questions. First, how is amino acid availability sensed to regulate lysosomal abundance of FLCN? Second, what is the relationship between FLCN lysosome localization, RagA/B interactions, and RagC/D GAP activity? In this study, we show that RagA/B nucleotide status determines the FLCN–FNIP1 recruitment to lysosomes. Starvation-induced FLCN–FNIP lysosome localization requires GAP activity toward Rags 1 (GATOR1), the GAP that converts RagA/B to the guanosine diphosphate (GDP)-bound state. This places FLCN–FNIP recruitment to lysosomes under the control of amino acid sensors that act upstream of GATOR1. By binding to RagA/BGDP and acting on RagC/D, FLCN–FNIP can coordinate nucleotide status between Rag heterodimer subunits in response to changes in amino acid availability.
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Shen, Kuang, and David M. Sabatini. "Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms." Proceedings of the National Academy of Sciences 115, no. 38 (September 4, 2018): 9545–50. http://dx.doi.org/10.1073/pnas.1811727115.
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The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique “locking” mechanism to stabilize its active (GTPRagA–RagCGDP) or inactive (GDPRagA–RagCGTP) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency.
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Lypaczewski, Patrick, Wen-Wei Zhang, and Greg Matlashewski. "Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence." PLOS Neglected Tropical Diseases 15, no. 2 (February 23, 2021): e0009079. http://dx.doi.org/10.1371/journal.pntd.0009079.
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Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.
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Anandapadamanaban, Madhanagopal, Glenn R. Masson, Olga Perisic, Alex Berndt, Jonathan Kaufman, Chris M. Johnson, Balaji Santhanam, Kacper B. Rogala, David M. Sabatini, and Roger L. Williams. "Architecture of human Rag GTPase heterodimers and their complex with mTORC1." Science 366, no. 6462 (October 10, 2019): 203–10. http://dx.doi.org/10.1126/science.aax3939.
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The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
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Li, Kristina, Shogo Wada, Bridget S. Gosis, Chelsea Thorsheim, Paige Loose, and Zolt Arany. "Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3." PLOS Biology 20, no. 3 (March 31, 2022): e3001594. http://dx.doi.org/10.1371/journal.pbio.3001594.
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Mechanistic target of rapamycin complex I (mTORC1) is central to cellular metabolic regulation. mTORC1 phosphorylates a myriad of substrates, but how different substrate specificity is conferred on mTORC1 by different conditions remains poorly defined. Here, we show how loss of the mTORC1 regulator folliculin (FLCN) renders mTORC1 specifically incompetent to phosphorylate TFE3, a master regulator of lysosome biogenesis, without affecting phosphorylation of other canonical mTORC1 substrates, such as S6 kinase. FLCN is a GTPase-activating protein (GAP) for RagC, a component of the mTORC1 amino acid (AA) sensing pathway, and we show that active RagC is necessary and sufficient to recruit TFE3 onto the lysosomal surface, allowing subsequent phosphorylation of TFE3 by mTORC1. Active mutants of RagC, but not of RagA, rescue both phosphorylation and lysosomal recruitment of TFE3 in the absence of FLCN. These data thus advance the paradigm that mTORC1 substrate specificity is in part conferred by direct recruitment of substrates to the subcellular compartments where mTORC1 resides and identify potential targets for specific modulation of specific branches of the mTOR pathway.
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Rogala, Kacper B., Xin Gu, Jibril F. Kedir, Monther Abu-Remaileh, Laura F. Bianchi, Alexia M. S. Bottino, Rikke Dueholm, et al. "Structural basis for the docking of mTORC1 on the lysosomal surface." Science 366, no. 6464 (October 10, 2019): 468–75. http://dx.doi.org/10.1126/science.aay0166.
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The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.
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Alhazmi, Hatem, Syyed Shah, and Atif Mahmood. "Sustainable Development of Innovative Green Construction Materials: A Study for Economical Eco-Friendly Recycled Aggregate Based Geopolymer Concrete." Materials 13, no. 21 (October 30, 2020): 4881. http://dx.doi.org/10.3390/ma13214881.
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Green revolution and high carbon footprint concepts have attracted the development of a green and sustainable environment. This work endeavors to investigate the behavior of recycled aggregate geopolymer concrete (RAGC) developed with four different types of effluents to develop sustainability in the construction industry and to produce an eco-friendly environment. Each of the types of effluents was used by completely replacing the freshwater in RAGC to examine its influence on compressive strength (CS), chloride ion migration (CIM), split tensile strength (STS), and resistance to the sulfuric acid attack of RAGC at various testing ages. The test outputs portray that the effluent obtained from the textile mill performed well for the CS (25% higher than the control mix) and STS (17% higher than the control mix) of RAGC. Similarly, the highest mass loss of RAGC due to the acid attack (41% higher than control mix) and the highest CIM (29% higher than control mix) were represented by the RAGC mix made with effluent obtained from fertilizer mill. The statistical analysis indicated no significant influence of using textile mill effluent (TE), fertilizer mill effluent (FE), and sugar mill effluent (SE) on the STS, CIM, and mass loss due to acid attack while it presented a significant influence on the CS of various mixes. Therefore, this investigation solidly substantiates the acceptability of studied types of effluents for the fabrication of eco-friendly green materials.
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Lawrence, Rosalie E., Simon A. Fromm, Yangxue Fu, Adam L. Yokom, Do Jin Kim, Ashley M. Thelen, Lindsey N. Young, et al. "Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex." Science 366, no. 6468 (October 31, 2019): 971–77. http://dx.doi.org/10.1126/science.aax0364.
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The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo–electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen, and M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, no. 7 (July 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882-3889.1993.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen, and M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, no. 7 (July 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Suryawan, Agus, and Teresa A. Davis. "Amino Acid- and Insulin-Induced Activation of mTORC1 in Neonatal Piglet Skeletal Muscle Involves Sestrin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR Complex Formation." Journal of Nutrition 148, no. 6 (May 23, 2018): 825–33. http://dx.doi.org/10.1093/jn/nxy044.
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Abstract Background Feeding stimulates protein synthesis in skeletal muscle of neonates and this response is regulated through activation of mechanistic target of rapamycin complex 1 (mTORC1). The identity of signaling components that regulate mTORC1 activation in neonatal muscle has not been fully elucidated. Objective We investigated the independent effects of the rise in amino acids (AAs) and insulin after a meal on the abundance and activation of potential regulators of mTORC1 in muscle and whether the responses are modified by development. Methods Overnight-fasted 6- and 26-d-old pigs were infused for 2 h with saline (control group) or with a balanced AA mixture (AA group) or insulin (INS group) to achieve fed levels while insulin or AAs, respectively, and glucose were maintained at fasting levels. Muscles were analyzed for potential mTORC1 regulatory mechanisms and results were analyzed by 2-factor ANOVA followed by Tukey's post hoc test. Results The abundances of DEP domain-containing mTOR-interacting protein (DEPTOR), growth factor receptor bound protein 10 (GRB10), and regulated in development and DNA damage response 2 (REDD2) were lower (65%, 73%, and 53%, respectively; P < 0.05) and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1/2 (LAMTOR1/2), vacuolar H+-ATPase (V-ATPase), and Sestrin2 were higher (94%, 141%, 145%, and 127%, respectively; P < 0.05) in 6- than in 26-d-old pigs. Both AA and INS groups increased phosphorylation of GRB10 (P < 0.05) compared with control in 26- but not in 6-d-old pigs. Formation of Ras-related GTP-binding protein A (RagA)-mTOR, RagC-mTOR, and Ras homolog enriched in brain (RHEB)-mTOR complexes was increased (P < 0.05) and Sestrin2-GTPase activating protein activity towards Rags 2 (GATOR2) complex was decreased (P < 0.05) by both AA and INS groups and these responses were greater (P < 0.05) in 6- than in 26-d-old pigs. Conclusion The results suggest that formation of RagA-mTOR, RagC-mTOR, RHEB-mTOR, and Sestrin2-GATOR2 complexes may be involved in the AA- and INS-induced activation of mTORC1 in skeletal muscle of neonates after a meal and that enhanced activation of the mTORC1 signaling pathway in neonatal muscle is in part due to regulation by DEPTOR, GRB10, REDD2, LAMTOR1/2, V-ATPase, and Sestrin2.
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Davis, Teresa A., Samer El-Kadi, Agus Suryawan, and Marta Fiorotto. "356 Meal feeding compared with continuous feeding enhances insulin and amino acid signaling to translation initiation in skeletal muscle of pigs." Journal of Animal Science 97, Supplement_3 (December 2019): 127–28. http://dx.doi.org/10.1093/jas/skz258.261.
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Abstract Objectives: Meal feeding enhances skeletal muscle protein synthesis and lean growth more than continuous feeding in piglets. This enhanced muscle protein synthesis with meal feeding is associated with increased activation of mTORC1-dependent translation initiation. The mechanism underlying this response is unknown. We aimed to identify insulin and amino acid signaling components involved in the enhanced lean growth that results from meal feeding vs. continuous feeding in term-born pigs. Methods: Newborn piglets were fed for 21 d an equal amount of sow milk replacer (12.8 g protein and 155 kcal/(kg BW.d)) by gastrostomy tube either as intermittent bolus meals every 4 h (MEAL) or by continuous infusion (CON). After 21 d, gastrocnemius muscle was collected from CON, and before (MEAL-0) or 60 min after a meal (MEAL-60). Components of the insulin and amino acid signaling pathways up- and downstream of mTORC1 that regulate protein translation were measured. Results: Phosphorylation of AKT and TCS2 was greater in MEAL-60 than in MEAL-0 and CON (P < 0.05). The association of Sestrin2 with GATOR2 was similar in CON and MEAL-0 but was lower in MEAL-60 (P < 0.05). The abundances of RagA-mTOR, RagC-mTOR, and Rheb-mTOR, but not CASTOR1-GATOR2, complexes were higher in MEAL-60 than in CON and MEAL-0 (P < 0.05). The phosphorylation of S6K1 and 4EBP1 was higher in MEAL-60 than CON and MEAL-0 (P < 0.05). The abundances of Sestrin2, GATOR2, CASTOR1, RagA, RagC, and Rheb and the phosphorylation of eIF2alpha, eEF2, ERK1/2 and AMPK were unaffected by treatments. Conclusions: Our results demonstrate that the enhanced rate of skeletal muscle protein synthesis and lean growth with meal feeding compared with continuous feeding are due to the enhanced activation of both insulin and amino acid signaling pathways that result in the greater stimulation of translation initiation. Support: NIH HD085573, USDA CRIS 6250-51000-055, NIH HD072891, USDA NIFA 2013-67015-20438.
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Boutry, Claire, Samer W. El-Kadi, Agus Suryawan, Julia Steinhoff-Wagner, Barbara Stoll, Renán A. Orellana, Hanh V. Nguyen, Scot R. Kimball, Marta L. Fiorotto, and Teresa A. Davis. "Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs." American Journal of Physiology-Endocrinology and Metabolism 310, no. 8 (April 15, 2016): E699—E713. http://dx.doi.org/10.1152/ajpendo.00479.2015.
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Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets ( n = 11–12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 μmol·kg−1·h−1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.
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Dudley, Darryll D., JoAnn Sekiguchi, Chengming Zhu, Moshe J. Sadofsky, Scott Whitlow, Jeffrey DeVido, Robert J. Monroe, Craig H. Bassing, and Frederick W. Alt. "Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice." Journal of Experimental Medicine 198, no. 9 (October 27, 2003): 1439–50. http://dx.doi.org/10.1084/jem.20030627.
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RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.
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Nada, Shigeyuki, and Masato Okada. "Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1." Journal of Biochemistry 168, no. 6 (July 11, 2020): 621–32. http://dx.doi.org/10.1093/jb/mvaa076.
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Abstract Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.
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Suryawan, Agus, Marko Rudar, Marta L. Fiorotto, and Teresa A. Davis. "Differential regulation of mTORC1 activation by leucine and β-hydroxy-β-methylbutyrate in skeletal muscle of neonatal pigs." Journal of Applied Physiology 128, no. 2 (February 1, 2020): 286–95. http://dx.doi.org/10.1152/japplphysiol.00332.2019.
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Leucine (Leu) and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent protein synthesis in the skeletal muscle of neonatal pigs. This study aimed to determine whether HMB and Leu utilize common nutrient-sensing mechanisms to activate mTORC1. In study 1, neonatal pigs were fed one of five diets for 24 h: low protein (LP), high protein (HP), or LP supplemented with 4 (LP+HMB4), 40 (LP+HMB40), or 80 (LP+HMB80) μmol HMB·kg body wt−1·day−1. In study 2, neonatal pigs were fed for 24 h: LP, LP supplemented with Leu (LP+Leu), or HP diets delivering 9, 18, and 18 mmol Leu·kg body wt−1·day−1, respectively. The upstream signaling molecules that regulate mTORC1 activity were analyzed. mTOR phosphorylation on Ser2448 and Ser2481 was greater in LP+HMB40, LP+HMB80, and LP+Leu than in LP and greater in HP than in HMB-supplemented groups ( P < 0.05), whereas HP and LP+Leu were similar. Rheb-mTOR complex formation was lower in LP than in HP ( P < 0.05), with no enhancement by HMB or Leu supplementation. The Sestrin2-GATOR2 complex was more abundant in LP than in HP and was reduced by Leu ( P < 0.05) but not HMB supplementation. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than in LP and HMB groups ( P < 0.05). There were no treatment differences in RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex abundances. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was lower in LP than HP ( P < 0.05) and unaffected by HMB or Leu supplementation. Our results demonstrate that HMB stimulates mTORC1 activation in neonatal muscle independent of the leucine-sensing pathway mediated by Sestrin2 and the Rag proteins. NEW & NOTEWORTHY Dietary supplementation with either leucine or its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulates protein synthesis in skeletal muscle of the neonatal pig. Our results demonstrate that both leucine and HMB stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) phosphorylation in neonatal muscle. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased binding of Rag A/C to mTOR. However, HMB’s activation of mTORC1 is independent of this leucine-sensing pathway.
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Kimball, Scot R., Suhana Ravi, Bradley S. Gordon, Michael D. Dennis, and Leonard S. Jefferson. "Amino Acid–Induced Activation of mTORC1 in Rat Liver Is Attenuated by Short-Term Consumption of a High-Fat Diet." Journal of Nutrition 145, no. 11 (September 23, 2015): 2496–502. http://dx.doi.org/10.3945/jn.115.215491.
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Abstract Background: The chronic activation of the mechanistic (mammalian) target of rapamycin in complex 1 (mTORC1) in response to excess nutrients contributes to obesity-associated pathologies. Objective: To understand the initial events that ultimately lead to obesity-associated pathologies, the present study assessed mTORC1 responses in the liver after a relatively short exposure to a high-fat diet (HFD). Methods: Male, obesity-prone rats were meal-trained to consume either a control (CON; 10% of energy from fat) diet or an HFD (60% of energy from fat) for 2 wk. Livers were collected and analyzed for mTORC1 signaling [assessed by changes in phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1)] and potential regulatory mechanisms, including changes in the association of Ras-related GTP binding (Rag) A and RagC with mechanistic target of rapamycin (mTOR) and expression of Sestrin1, Sestrin2, and Sestrin3. Results: Feeding-induced activation of mTORC1 was blunted in the livers of rats fed the HFD compared with those fed the CON diet (p70S6K1 phosphorylation, 19% of CON; 4E-BP1 phosphorylation, 61% of CON). The attenuated response was not due to a change in a kinase also referred to as protein kinase B (Akt) signaling but rather to resistance to amino acid–induced activation of mTORC1, as evidenced by a reduction in the interaction of RagA (69% of CON) and RagC (66% of CON) with mTOR and enhanced expression of the mTORC1 repressors Sestrin2 (132% of CON) and Sestrin3 (143% of CON). The consumption of an HFD led to impaired amino acid–induced activation of mTORC1 as assessed in livers perfused in situ with medium containing various concentrations of amino acids. Conclusions: These results in rats support a model in which the initial response of the liver to an HFD is an attenuation of, rather than the expected activation of, mTORC1. The initial response likely represents a counterregulatory mechanism to handle the onset of excess nutrients and is caused by enhanced expression of Sestrin2 and Sestrin3, which, in turn, leads to impaired Rag signaling, resulting in resistance to amino acid–induced activation of mTORC1.
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Nilavar, Namrata M., Mayilaadumveettil Nishana, Amita M. Paranjape, Raghunandan Mahadeva, Rupa Kumari, Bibha Choudhary, and Sathees C. Raghavan. "Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs." Biochemical Journal 477, no. 18 (September 24, 2020): 3567–82. http://dx.doi.org/10.1042/bcj20200361.
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Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
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Alashker, Yasser, and Ali Raza. "Seismic Performance of Recycled Aggregate Geopolymer Concrete-Filled Double Skin Tubular Columns with Internal Steel and External FRP Tube." Polymers 14, no. 23 (November 29, 2022): 5204. http://dx.doi.org/10.3390/polym14235204.
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The large production of cement is resulting in a high-carbon footprint, which is essential to minimize for sustainable concrete construction. Moreover, the large quantity of recycled coarse aggregate (RCA) from the demolition of old concrete structures is creating problems for landfill and disposal. The primary goal of this study is to investigate the seismic efficiency of innovative fiber-reinforced polymer (FRP)-recycled aggregate geopolymer concrete (RAGC) steel-tubed columns (FGSTCs) with an internal steel tube (STT), an external glass-FRP tube (GLT), and RAGC located between the two-tubed components to develop a serviceable structural element. To study their seismic functioning under axial load and lateral repeated load, five FGSTC specimens were manufactured and analyzed under quasi-static loads. The influence of three variables on the performance of FGSTC specimens, consisting of STT reinforcing ratio, compression ratio, and recycled coarse aggregate (RCA) replacement ratio, was investigated in this investigation. The produced specimens’ ductility, hysteretic loops, strain distribution, skeleton curves, stiffness functioning, energy capacity dissipation, damaging functioning, and strength loss were all assessed and discussed. The results of this investigation revealed that percentage substitution of RCA had a minor impact on the seismic functioning of FGSTCs; however, the compression-load ratio depicted a substantial impact. The energy loss of the FGSTCs was 24.5% higher than that of their natural aggregate equivalents. FGSTCs may have a 16.9% lower cumulative failure rate than their natural aggregate counterparts.
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Hnatova, Martina, Micheline Wésolowski-Louvel, Guenaëlle Dieppois, Julien Deffaud, and Marc Lemaire. "Characterization of KlGRR1 and SMS1 Genes, Two New Elements of the Glucose Signaling Pathway of Kluyveromyces lactis." Eukaryotic Cell 7, no. 8 (June 13, 2008): 1299–308. http://dx.doi.org/10.1128/ec.00454-07.
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ABSTRACT The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Δsms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Δsms1 mutation.
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Swaminathan, Srividya, Lars Klemm, Eugene Park, Anthony M. Ford, Soo-mi Kweon, Daniel Trageser, Brian Hasselfeld, et al. "Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 861. http://dx.doi.org/10.1182/blood.v124.21.861.861.
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Abstract Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia. Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion. Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions. Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. Disclosures No relevant conflicts of interest to declare.
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Akamatsu, Yoshiko, and Marjorie A. Oettinger. "Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4670–78. http://dx.doi.org/10.1128/mcb.18.8.4670.
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ABSTRACT The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.
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Wu, Zeguang, Narmadha Subramanian, Eva-Maria Jacobsen, Kerstin Laib Sampaio, Johannes van der Merwe, Manfred Hönig, and Thomas Mertens. "NK Cells from RAG- or DCLRE1C-Deficient Patients Inhibit HCMV." Microorganisms 7, no. 11 (November 10, 2019): 546. http://dx.doi.org/10.3390/microorganisms7110546.
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The recombination-activating genes (RAGs) and the DNA cross-link repair 1C gene (DCLRE1C) encode the enzymes RAG1, RAG2 and Artemis. They are critical components of the V(D)J recombination machinery. V(D)J recombination is well known as a prerequisite for the development and antigen diversity of T and B cells. New findings suggested that RAG deficiency impacts the cellular fitness and function of murine NK cells. It is not known whether NK cells from severe combined immunodeficiency (SCID) patients with defective RAGs or DCLRE1C (RAGs−/DCLRE1C−-NK) are active against virus infections. Here, we evaluated the anti-HCMV activity of RAGs−/DCLRE1C−-NK cells. NK cells from six SCID patients were functional in inhibiting HCMV transmission between cells in vitro. We also investigated the expansion of HCMV-induced NK cell subset in the RAG- or DCLRE1C-deficient patients. A dynamic expansion of NKG2C+ NK cells in one RAG-2-deficient patient was observed post HCMV acute infection. Our study firstly reveals the antiviral activity of human RAGs−/ DCLRE1C−-NK cells.
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Shi, Xiaoju, Shirley A. Hanley, Marie-Claire Faray-Kele, Stuart C. Fawell, Joseph Aduse-Opoku, Robert A. Whiley, Michael A. Curtis, and Lucinda M. C. Hall. "The rag Locus of Porphyromonas gingivalis Contributes to Virulence in a Murine Model of Soft Tissue Destruction." Infection and Immunity 75, no. 4 (February 5, 2007): 2071–74. http://dx.doi.org/10.1128/iai.01785-06.
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ABSTRACT The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.
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Naberhuis, Jane K., Agus Suryawan, Hanh V. Nguyen, Adriana Hernandez-Garcia, Stephanie M. Cruz, Patricio E. Lau, Oluyinka O. Olutoye, et al. "Prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of neonatal piglets." American Journal of Physiology-Endocrinology and Metabolism 317, no. 5 (November 1, 2019): E839—E851. http://dx.doi.org/10.1152/ajpendo.00151.2019.
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Postnatal growth of lean mass is commonly blunted in preterm infants and may contribute to short- and long-term morbidities. To determine whether preterm birth alters the protein anabolic response to feeding, piglets were delivered at term or preterm, and fractional protein synthesis rates (Ks) were measured at 3 days of age while fasted or after an enteral meal. Activation of signaling pathways that regulate protein synthesis and degradation were determined. Relative body weight gain was lower in preterm than in term. Gestational age at birth (GAB) did not alter fasting plasma glucose or insulin, but when fed, plasma insulin and glucose rose more slowly, and reached peak value later, in preterm than in term. Feeding increased Ks in longissimus dorsi (LD) and gastrocnemius muscles, heart, pancreas, and kidney in both GAB groups, but the response was blunted in preterm. In diaphragm, lung, jejunum, and brain, feeding increased Ks regardless of GAB. Liver Ks was greater in preterm than term and increased with feeding regardless of GAB. In all tissues, changes in 4EBP1, S6K1, and PKB phosphorylation paralleled changes in Ks. In LD, eIF4E·eIF4G complex formation, phosphorylation of TSC2, mTOR, and rpS6, and association of mammalian target of rapamycin (mTOR1) complex with RagA, RagC, and Rheb were increased by feeding and blunted by prematurity. There were no differences among groups in LD protein degradation markers. Our results demonstrate that preterm birth reduces weight gain and the protein synthetic response to feeding in muscle, pancreas, and kidney, and this is associated with blunted insulin- and/or amino acid-induced translation initiation signaling.
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Meru, Nadine, Andreas Jung, Irith Baumann, and Gerald Niedobitek. "Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils." Blood 99, no. 2 (January 15, 2002): 531–37. http://dx.doi.org/10.1182/blood.v99.2.531.
Full textAbstract:
Abstract V(D)J recombination in lymphocytes is mediated by 2 recombination-activating genes, RAG1 and RAG2,which are expressed during lymphocyte development in bone marrow and thymus. Prompted by studies reporting re-expression of the RAGs in germinal center B cells, the expression of RAGs and terminal deoxynucleotidyl transferase (TdT) in human lymphoid tissues was examined using in situ hybridization and immunohistochemistry, respectively. Here it is shown that RAGs and TdT are not reinduced in germinal center reactions. However, RAG+/TdT+ cells are frequently present in extrafollicular areas of tonsils mainly at the boundary between lymphoid tissue and fibrous scaffold. Phenotypic analyses suggest that these cells are B cells. Finally, it is shown that RAG+/TdT+ cells are found more frequently in tonsils than in other peripheral lymphoid tissues. This may reflect an increased influx of RAG+/TdT+ cells as a result of higher antigenic stimulation at this site. Alternatively, this observation may indicate that the tonsils are an additional site of lymphocyte ontogeny.
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Nagano, Keiji, Yukitaka Murakami, Kiyoshi Nishikawa, Junpei Sakakibara, Kazuo Shimozato, and Fuminobu Yoshimura. "Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants." Journal of Medical Microbiology 56, no. 11 (November 1, 2007): 1536–48. http://dx.doi.org/10.1099/jmm.0.47289-0.
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The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the ΔragA mutant reduced RagB expression considerably and the ΔragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the ΔragA and ΔragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the ΔragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein–protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.
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McMahan, C. J., M. J. Sadofsky, and D. G. Schatz. "Definition of a large region of RAG1 that is important for coimmunoprecipitation of RAG2." Journal of Immunology 158, no. 5 (March 1, 1997): 2202–10. http://dx.doi.org/10.4049/jimmunol.158.5.2202.
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Abstract Interaction between the RAG1 and RAG2 proteins is probably critical for V(D)J recombination. Using a coimmunoprecipitation assay, we define a large region of RAG1 (amino acids 504-1008) that is sufficient for interaction with RAG2. This region comprises the C-terminal half of the RAG1 protein, and is within the region defined as the recombinationally active core. Deletion of either of two regions of RAG1 (amino acids 504-570 or 850-1008) causes a loss of interaction with RAG2. Loss of coimmunoprecipitation is also seen with RAG1 core proteins containing deletions of smaller stretches of amino acids (amino acids 506-511 or 545-550), emphasizing the importance of this region of RAG1 in forming a complex with RAG2. A variety of other small deletion mutations within the amino acid region 504-1008 also decrease coimmunoprecipitation of RAG2 with RAG1, indicating that much or all of this region is important for complex formation.
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Magalhães, Joana, and Vanessa Valdiglesias. "Análisis de la brecha de género en los premios otorgados por la Real Academia Galega de Ciencias (RAGC)." Cuestiones de género: de la igualdad y la diferencia, no. 17 (June 30, 2022): 12–31. http://dx.doi.org/10.18002/cg.i17.7294.
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A pesar de los importantes avances en cuanto a la participación de las mujeres en el desarrollo científico y tecnológico en nuestro país, persiste una clara infrarrepresentación femenina en las academias de ciencias e ingeniería. La brecha de género es también visible en los galardones otorgados por estas instituciones. En este trabajo se analiza la brecha de género en los premios otorgados por la Real Academia Galega de Ciencias (RAGC) en 30 de sus convocatorias en el periodo 2013-2021, enfocándonos en varios aspectos relevantes: la composición de los jurados, las candidaturas presentadas y las personas finalmente galardonadas. En la mayoría de los galardones analizados se pone de manifiesto un claro sesgo de género a favor de los varones que se va incrementando a medida que aumenta la cuantía del premio o la relevancia del galardón, con la consiguiente repercusión social en la valoración y visibilización del trabajo de las científicas.
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Kondratenko, I. V., O. E. Pashchenko, Y. A. Rodina, M. V. Belevtcev, Den M. Van, and A. A. Bologov. "Clinical and laboratory phenotypes of severe combined immunodeficiencies with mutations in RAG1/RAG2 genes." Russian Journal of Allergy 9, no. 4 (December 15, 2012): 26–32. http://dx.doi.org/10.36691/rja689.
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Background. The RAG1 and RAG2 proteins are key players in the V(D)J recombination process leading to the assembly of antigen receptor genes. Defects in RAG1/RAG2 genes are caused to different phenotypes of severe combined immunodeficiencies (SCID). Objective — to determine the clinical and laboratory manifestations in patients with RAG1 / RAG2 mutations from one single center, to identify the phenotype-genotype correlations. Materials and methods. We described 4 children with RAG1 mutations. Diagnosis of SCID was confirmed by criteria’s of European society of immunodeficiencies (ESID). Results. In two patients we observed Omenn syndrome, in 1 — classic T -B -NK + SCID, in 1 — «sof» T +B -NK + SCID. One patient with Omenn syndrome and patient with «soft» SCID had same RAG1 mutations. Conclusions. RAG 1 / RAG2 mutations are caused to severe life-threatening combined immunodeficiency, requiring radical therapy. We found no genotype-phenotype correlations in patients with RAG1 defects.
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Bailin, Tu, Xianming Mo, and Moshe J. Sadofsky. "A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination." Molecular and Cellular Biology 19, no. 7 (July 1, 1999): 4664–71. http://dx.doi.org/10.1128/mcb.19.7.4664.
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ABSTRACT During V(D)J recombination two proteins, RAG1 and RAG2, assemble as a protein-DNA complex with the appropriate DNA targets containing recombination signal sequences (RSSs). The properties of this complex require a fairly elaborate set of protein-protein and protein-DNA contacts. Here we show that a purified derivative of RAG1, without DNA, exists predominantly as a homodimer. A RAG2 derivative alone has monomer, dimer, and larger forms. The coexpressed RAG1 and RAG2 proteins form a mixed tetramer in solution which contains two molecules of each protein. The same tetramer of RAG1 and RAG2 plus one DNA molecule is the form active in cleavage. Additionally, we show that both DNA products following cleavage can still be held together in a stable protein-DNA complex.
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Zarrin, A. A., I. Fong, L. Malkin, P. A. Marsden, and N. L. Berinstein. "Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions." Journal of Immunology 159, no. 9 (November 1, 1997): 4382–94. http://dx.doi.org/10.4049/jimmunol.159.9.4382.
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Abstract Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.
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Laufenberg, Lacee J., Kristen T. Crowell, and Charles H. Lang. "Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle." Nutrients 13, no. 4 (April 9, 2021): 1236. http://dx.doi.org/10.3390/nu13041236.
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The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol’s effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein–protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min (“refed”) or remained food-deprived (“fasted”). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein–protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.
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Naik, Abani Kanta, Aaron T. Byrd, Aaron C. K. Lucander, and Michael S. Krangel. "Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes." Journal of Experimental Medicine 216, no. 1 (December 13, 2018): 231–43. http://dx.doi.org/10.1084/jem.20181402.
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Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.
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Klemm, Lars, Srividya Swaminathan, Elli Papaemmanuil, Anthony M. Ford, Mel Greaves, Rafael Casellas, David Schatz, Michael R. Lieber, and Markus Muschen. "Exposure to Inflammatory Immune Responses As Driver of Clonal Evolution in Childhood Acute Lymphoblastic Leukemia." Blood 126, no. 23 (December 3, 2015): 166. http://dx.doi.org/10.1182/blood.v126.23.166.166.
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Abstract Background: Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1 gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear. Rationale: Activation Induced Cytidine Deaminase (AID) and Recombination Activation Genes 1 and 2 (RAG1-RAG2) are genetic modifiers of the immunoglobulin (Ig) genes that are expressed during normal B cell development. Although AID and RAG1/RAG2 are thought to be segregated to early (RAG1/RAG2) and late (AID) stages of B cell development, respectively, we found that the two enzymes can be concurrently expressed during early B-lymphopoiesis in the context of repeated inflammatory stimuli. Results: Our experiments identified transitional pre-B cells as the subset that is particularly vulnerable to concomitant expression of AID and RAG1-RAG2 with earlier B cells being protected by IL7 signaling.Human B cells from children lacking a functional IL-7 receptor (IL-7R) displayed both increased expression and activity of AID concurrently with RAG1-RAG2. These results demonstrated that AID activation in both mouse and human early B cell compartments increases genetic instability. Although concurrent activation of AID and RAG1-RAG2 in patient samples implicated a correlation between the two enzymes in the pathogenesis of leukemia, this as such did not prove that the enzymes causally induce overt leukemogenesis. Therefore, we next evaluated the requirement of AID and RAG1-RAG2 in leukemogenic transformation, and identified a condition that leads to massive activation of these enzymes in a pre-leukemic B cell. Importantly, AID and RAG1-RAG2 expression increased dramatically during inflammatory immune responses (e.g. infection), where both these enzymes diversify the antibody repertoire and improve its affinity to antigens from infectious pathogens. We therefore tested whether the pre-B cell subset that concurrently expresses AID and RAG1-RAG2 can respond to an inflammatory stimulus, such as LPS. We observed that pre-B cells require protection from IL7, which prevents aberrant activation of AID. In the absence of protective IL-7, these pre-B cells acquired responsiveness to LPS and strongly activated AID concurrently with RAG1-RAG2 enzymes. We developed IL7-dependent pre-B cell cultures as a disease model for ETV6-RUNX1 pre-leukemia and tested the role of AID and RAG1 in the progression of pre-leukemic clones. To this end, we expanded ETV6-RUNX1 pre-B cells from wildtype (AID and RAG1 expressing) mice, or from mice lacking AID (Aid-/-Rag1+/+) or RAG1 (Aid+/+Rag1-/-). We then challenged pre-B cell cultures by withdrawal of IL7 (loss of protection) and inflammatory stimuli (LPS) and transplanted pre-B cells that had undergone five cycles of -IL7/LPS challenge. Upon transplanting -IL7/LPS-treated Aid+/+Rag1+/+ or Aid-/-Rag1+/+ or Aid+/+Rag1-/- pre-B cells containing ETV6-RUNX1 into NOD-SCID recipient mice, we observed that loss of either Aid or Rag1 dramatically prolonged the latency and reduced the penetrance of leukemia in transplant recipients. This proved that AID and RAG1-RAG2 causally accelerate clonal evolution of a pre-leukemic B cell towards leukemia. Our findings provide a mechanism by which pre-leukemic clones carrying a prenatal genetic lesion such as ETV6-RUNX1 can evolve through infectious and inflammatory stimuli ultimately leading to full blown leukemia. Conclusion: The impact of inflammatory stimuli on leukemogenesis has been previously implicated in multiple epidemiological studies. For instance, day-care attendance primed the immune system during early childhood and is thought to protect against exacerbation of B cell responses and to prevent collateral damage driving clonal evolution towards leukemia. Although inflammation (LPS stimulation) seems to play a role in accelerating pre-B leukemogenesis in our model, further experiments testing actual infectious pathogens are needed to corroborate this concept. Moreover, it is crucial to test whether leukemogenesis is accelerated in individuals infected with restricted classes of pathogens, not all of which may activate AID in pre-B cells. Disclosures No relevant conflicts of interest to declare.
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Malshetty, Vidyasagar, Jian Chen, Mary Hanna, and Patricia Cortes. "Role of Pax5 and YY1 in regulation of V(D)J recombination (111.1)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 111.1. http://dx.doi.org/10.4049/jimmunol.188.supp.111.1.
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Abstract The infinite variety of antigen-binding receptors originates from the rearrangement of B and T-cell receptor loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. Deregulation of this process, leads to a spectrum of diseases including immunodeficiency and leukemia. These leukemias are believed to arise from RAG1/2 mediated oncogenic chromosomal translocations. The transcription factor Pax5 is critical for B cell development and is known that, it interacts with RAG1/2 and mediates V to DJ recombination. Pax5 is also been implicated in human B cell malignancies. Another multifunctional transcription factor, YY1, is known to play a role in V to DJ recombination similar to Pax5. We were able to see an interaction of YY1 with RAG1/2 and Pax5, when these proteins were over-expressed in 293T cells. When we performed a plasmid based recombination assay in presence of Pax5, YY1 or both a consistent decrease in recombination levels was observed. To elucidate the mechanism of inhibition, we analyzed the recombination intermediates and found that the initial cleavage step is inhibited by these two factors. In addition, purified Pax5 and YY1 inhibit the in vitro cleavage activity of RAG1/2 recombinase. The expression of a mutant Pax5 increased the RAG recombinase activity, suggesting the importance of these two factors in regulation of RAGs mediated recombination.
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Sawchuk, Dennis J., Frances Weis-Garcia, Sohail Malik, Eva Besmer, Michael Bustin, Michel C. Nussenzweig, and Patricia Cortes. "V(D)J Recombination: Modulation of RAG1 and RAG2 Cleavage Activity on 12/23 Substrates by Whole Cell Extract and DNA-bending Proteins." Journal of Experimental Medicine 185, no. 11 (June 2, 1997): 2025–32. http://dx.doi.org/10.1084/jem.185.11.2025.
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Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.
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Hao, Bingtao, Abani Kanta Naik, Akiko Watanabe, Hirokazu Tanaka, Liang Chen, Hunter W. Richards, Motonari Kondo, et al. "An anti-silencer– and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development." Journal of Experimental Medicine 212, no. 5 (April 6, 2015): 809–24. http://dx.doi.org/10.1084/jem.20142207.
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Rag1 and Rag2 gene expression in CD4+CD8+ double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.
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Xue, Jeff, Kelly Keys, Aaron McCoy, and Edward Weinstein. "Rag1 and Rag2 Knockout rats to drive xenografts and oncology studies (126.30)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 126.30. http://dx.doi.org/10.4049/jimmunol.188.supp.126.30.
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Abstract Rejection of grafted tissues by host immune systems is problematic during xenotransplantation. To help alleviate this and provide models amenable to the xenograft process, we created rats with genetic modifications of genes: Rag1 or Rag2. Rag1 and Rag2 play an important role in the rearrangement and recombination of genes of immunoglobulin and T-cell receptor molecules during the process of VDJ recombination. These rats were created via zinc finger nuclease (ZFN) targeted editing of Sprague-Dawley and F344 rat genomes. ZFN creates a double stranded break at the target locus. This lesion is repaired by the low fidelity repair mechanism non-homologous end-joining which commonly yields out of frame mutations in the gene, leading to non-functional proteins. Phenotypic characterization of these animals occurs when homozygosity is achieved. Rag1 and Rag2 protein ablation was demonstrated by western blot analysis. Flow cytometric analysis of peripheral blood cells in Rag1 KO animals at 4, 10 and 20 weeks demonstrated a significant decrease in mature T and B lymphocytes relative to age-matched wild type rats. Rag2 KO rats demonstrate a non-leaky depletion of mature T and B lymphocytes at 4 and 10 weeks. By employing the ZFN technique, we developed Rag1 and Rag2 KO rats with compromised immunology systems, which we believe will be helpful in the studies of xenografts, cancer, vaccine development, autoimmune, infectious and GVHD diseases.
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Eastman, Quinn M., Isabelle J. Villey, and David G. Schatz. "Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3788–97. http://dx.doi.org/10.1128/mcb.19.5.3788.
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ABSTRACT V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.
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De, Pallabi, Mandy M. Peak, and Karla K. Rodgers. "DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated." Molecular and Cellular Biology 24, no. 15 (August 1, 2004): 6850–60. http://dx.doi.org/10.1128/mcb.24.15.6850-6860.2004.
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ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.
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Fisher, Megan, and Craig Bassing. "Pre-B cells suppress RAG expression in response to DNA double-strand breaks (HEM1P.225)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 50.8. http://dx.doi.org/10.4049/jimmunol.194.supp.50.8.
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Abstract The ability of the Rag1/Rag2 (RAG) endonuclease to assemble antigen receptor (AgR) genes is essential for adaptive immunity. However, aberrant induction or repair of RAG-induced DNA double strand breaks (DSBs) can lead to oncogenic AgR translocations. We have previously shown that RAG-induced DSBs in pre-B cells activate the ATM kinase to prevent RAG cleavage of the homologous allele, and to inhibit expression of Rag1 and Rag2. These breaks also suppress expression of Gadd45α, which promotes Rag1 and Rag2 transcription. Since DSBs induced by ionizing radiation (IR) signal through ATM to increase Gadd45α expression in all other cell types including mature B cells, these results indicate that RAG DSBs and/or pre-B cells use unique mechanisms to regulate Gadd45α expression. We now demonstrate that IR-induced DSBs in pre-B cells signal through the ATM kinase to down-regulate Gadd45α expression. We also demonstrate that these IR DSBs signal ATM-dependent suppression of Rag1 and Rag2 transcription, without affecting stability of Rag1 or Rag2 mRNA. Because the presence of multiple DSBs in a cell greatly increases the risk of translocation, we hypothesize that pre-B cells have developed a unique developmental stage-specific DSB response to suppress the induction of RAG DSBs in the presence of other DSBs. Our ongoing studies will determine the role of this pre-B cell specific DSB response in suppressing AgR translocations and enforcing mono-allelic assembly of immunoglobulin genes.
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Gomez, Carlos A., Leon M. Ptaszek, Anna Villa, Fabio Bozzi, Cristina Sobacchi, Edward G. Brooks, Luigi D. Notarangelo, et al. "Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies." Molecular and Cellular Biology 20, no. 15 (August 1, 2000): 5653–64. http://dx.doi.org/10.1128/mcb.20.15.5653-5664.2000.
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ABSTRACT The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and ΔI273 within the predicted second β-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second β-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.
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Tsun, Zhi-Yang, Liron Bar-Peled, Lynne Chantranupong, Roberto Zoncu, Tim Wang, Choah Kim, Eric Spooner, and David M. Sabatini. "The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTORC1." Molecular Cell 52, no. 4 (November 2013): 495–505. http://dx.doi.org/10.1016/j.molcel.2013.09.016.
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Hegedűs, Krisztina, Péter Nagy, Zoltán Gáspári, and Gábor Juhász. "The Putative HORMA Domain Protein Atg101 Dimerizes and Is Required for Starvation-Induced and Selective Autophagy inDrosophila." BioMed Research International 2014 (2014): 1–13. http://dx.doi.org/10.1155/2014/470482.
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The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy inDrosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2)P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show thatDrosophilaAtg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2)P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.
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Raval, Prafulla, Sushil Kumar, Aleksei N. Kriatchko, and Patrick C. Swanson. "Evidence for Ku70/Ku80 association with full length RAG1 (35.6)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S2. http://dx.doi.org/10.4049/jimmunol.178.supp.35.6.
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Abstract Antigen receptor genes are assembled by a form of site-specific DNA rearrangement termed V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at recombination signal sequences (RSSs), and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present biochemical evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1 and can become integrated into a stable RAG-RSS complex assembled with full-length RAG1 and core RAG2, but not core RAG1 (aa 384–1040) and either core or full-length RAG2. Formation of this complex minimally requires residues 211–1040 of RAG1. These results provide a biochemical link between the two phases of V(D)J recombination. This research is supported by National Institutes of Health grant 1R01 AI055599 to P.C.S.
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Kim, Maengjo, Linghui Lu, Alexey V. Dvornikov, Xiao Ma, Yonghe Ding, Ping Zhu, Timothy M. Olson, Xueying Lin, and Xiaolei Xu. "TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy." International Journal of Molecular Sciences 22, no. 11 (May 23, 2021): 5494. http://dx.doi.org/10.3390/ijms22115494.
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A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.