Structural mechanism for amino acid-dependent Rag GTPase switching by SLC38A9

The mechanistic target of rapamycin complex 1 (mTORC1) couples cell growth to nutrient, energy and growth factor availability (1–3). mTORC1 is activated at the lysosomal membrane when amino acids are replete via the Rag guanosine triphosphatases (GTPases) (4–6). Rags exist in two stable states, an inactive (RagA/BGDP:RagC/DGTP) and active (RagA/BGTP:RagC/DGDP) state, during low and high cellular amino acid levels (4, 5). The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator (7, 8), and the FLCN:FNIP (FLCN-interacting protein) GTPase activating protein (GAP) complex (9), and prevents activation of the Rag dimer during amino acid starvation (10, 11). How the LFC is released upon amino acid refeeding is a major outstanding question in amino-acid dependent Rag activation. Here we show that the cytoplasmic tail of the lysosomal solute carrier family 38 member 9 (SLC38A9), a known Rag activator (12–14), destabilizes the LFC. By breaking up the LFC, SLC38A9 triggers the GAP activity of FLCN:FNIP toward RagC. We present the cryo electron microscopy (cryo-EM) structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre and post GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

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Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex

Science ◽

10.1126/science.aax0364 ◽

2019 ◽

Vol 366 (6468) ◽

pp. 971-977 ◽

Cited By ~ 27

Author(s):

Rosalie E. Lawrence ◽

Simon A. Fromm ◽

Yangxue Fu ◽

Adam L. Yokom ◽

Do Jin Kim ◽

...

Keyword(s):

Gtpase Activating Protein ◽

Interacting Protein ◽

Lysosomal Function ◽

Structural Mechanism ◽

Mechanistic Target Of Rapamycin ◽

Cryo Electron Microscopy ◽

Mtorc1 Signaling ◽

Gtpase Activation ◽

Guanosine Triphosphatase ◽

Rag Gtpase

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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Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9

Nature Structural & Molecular Biology ◽

10.1038/s41594-020-0490-9 ◽

2020 ◽

Vol 27 (11) ◽

pp. 1017-1023 ◽

Cited By ~ 1

Author(s):

Simon A. Fromm ◽

Rosalie E. Lawrence ◽

James H. Hurley

Keyword(s):

Amino Acid ◽

Structural Mechanism ◽

State Switching ◽

Rag Gtpase

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Faculty Opinions recommendation of Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738585253.793578657 ◽

2020 ◽

Author(s):

Qian Yin ◽

Bing Wang

Keyword(s):

Amino Acid ◽

Structural Mechanism ◽

State Switching ◽

Rag Gtpase

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Faculty Opinions recommendation of Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717956431.793467880 ◽

2012 ◽

Author(s):

Alfred Wittinghofer

Keyword(s):

Amino Acid ◽

Rag Gtpases ◽

Amino Acid Levels

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Faculty Opinions recommendation of The Rag GTPase Regulates the Dynamic Behavior of TSC Downstream of Both Amino Acid and Growth Factor Restriction .

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738621547.793578330 ◽

2020 ◽

Author(s):

Constantinos Demetriades

Keyword(s):

Amino Acid ◽

Growth Factor ◽

Dynamic Behavior ◽

Rag Gtpase

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Effects of amino acid levels during rearing on Cobb 500 slow-feathering broiler breeders: 1. Growth and development

Poultry Science ◽

10.1016/j.psj.2021.101327 ◽

2021 ◽

pp. 101327

Author(s):

Maria Camila Alfaro-Wisaquillo ◽

Edgar O. Oviedo-Rondón ◽

Hernan A. Cordova-Noboa ◽

Justina V. Caldas ◽

Gustavo A. Quintana-Ospina ◽

...

Keyword(s):

Amino Acid ◽

Growth And Development ◽

Broiler Breeders ◽

Amino Acid Levels

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88 Benefits of increasing amino acid intake in late gestation in prolific sows

Journal of Animal Science ◽

10.1093/jas/skaa054.136 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 76-76

Author(s):

Ron Ball ◽

Crystal L Levesque ◽

D J Cadogan

Keyword(s):

Birth Weight ◽

Amino Acid ◽

Reproductive Performance ◽

Essential Amino Acid ◽

Control Diet ◽

Genetic Selection ◽

Late Gestation ◽

Negative Effects ◽

Amino Acid Levels ◽

The Many

Abstract Most sows are fed a constant energy and amino acid supply throughout gestation, in line with the recommendations of most authorities and swine genetic companies. These recommendations for sow feeding have seen little change in decades, despite the many ways that sows have changed dramatically in reproductive performance. Beginning in about the year 2000, sow litter size has steadily increased as a result of genetic selection. With this increase in litter number has been a steady decline in birth weight, and the resulting negative effects of lower birthweight on subsequent piglet performance. Many experiments using so-called ‘bump’ feeding, or increased energy intake in late gestation, have been conducted in attempts to arrest this decline in birthweight and piglet performance. Generally, these experiments have shown little to no improvement in birthweight and often have negative effects on sow feed intake during gestation. These experiments have ignored the fact that the energy:amino acid ratios (lysine, threonine, isoleucine, tryptophan) in late gestation are different than during early and mid-gestation. In recent research in Australia we hypothesised that rapidly increasing essential amino acid levels in late gestation would increase birth weight and potentially improve subsequent reproductive performance. Three hundred and thirty-four multiparous PIC sows (average parity 3.6, average LW 261 kg) were housed in a dynamic gestation pen after mating and randomly assigned to one of two diet regimes. Two 13.5 MJ/kg DE gestation diets were formulated and created by blending in an ESF. The Control diet contained 0.48 g SID lysine per MJ DE and SID threonine, methionine+ cysteine, isoleucine and tryptophan at 68%, 65%, 58% and18% of SID lysine and offered at 2.2kg/day from d 28 to d 110. Sow were then moved to the farrowing house and placed on a lactation diet at 3.5kg/d. The Treatment diet contained 0.55 g SID lysine/MJ DE and SID threonine, methionine+cysteine, isoleucine and tryptophan at 78%, 65%, 60% and 20% of SID lysine and offered at 2.1kg/d from d 28 to d 85 and then increased to 2.4 kg/d to d 110 d. Increasing essential amino acid levels in late gestation increased gestational weight gain (5.6 kg, P=0.004), increased total litter birth weight (1.25 kg, P=0.003), and increased the birthweight of liveborn pigs from 1.286 to 1.329 kg, (P=0.04). There was no significant effect on the total number born or born alive. Piglet performance is not available because this commercial farm practices cross-fostering. Effects of continuation of this feeding regime in the same sows during subsequent parities is currently being evaluated.

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THE EFFECT OF GROWTH AND ADRENOCORTICOTROPIC HORMONES ON THE AMINO ACID LEVELS IN THE PLASMA

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)57061-7 ◽

1949 ◽

Vol 177 (1) ◽

pp. 91-95 ◽

Cited By ~ 7

Author(s):

Choh.Hao. Li ◽

I. Geschwind ◽

Herbert M. Evans

Keyword(s):

Amino Acid ◽

Amino Acid Levels

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Fetal sex modulates placental microRNA expression, potential microRNA-mRNA interactions, and levels of amino acid transporter expression and substrates: INFAT study subpopulation analysis of n-3 LCPUFA intervention during pregnancy and associations with offspring body composition

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00345-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Eva-Maria Sedlmeier ◽

Dorothy M. Meyer ◽

Lynne Stecher ◽

Manuela Sailer ◽

Hannelore Daniel ◽

...

Keyword(s):

Body Composition ◽

Amino Acid ◽

Amino Acid Transporter ◽

Microrna Expression ◽

Sexually Dimorphic ◽

Mrna Levels ◽

Control Male ◽

Cord Plasma ◽

Amino Acid Levels ◽

Acid Transporter

Abstract Background Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition. Results We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks’ gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age. Conclusions Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.

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