Structural basis for the docking of mTORC1 on the lysosomal surface

Rogala, Kacper B.; Gu, Xin; Kedir, Jibril Fetu; Abu-Remaileh, Monther; Bianchi, Laura F.; Bottino, Alexia M. S.; Dueholm, Rikke; Niehaus, Anna; Overwijn, Daan; Fils, Ange-Célia Priso; Zhou, Sherry; Leary, Daniel D.; Laqtom, Nouf N.; Brignole, Edward J.; Sabatini, David M.

doi:10.1126/science.aay0166

Cited by 141 publications

(185 citation statements)

References 79 publications

(54 reference statements)

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“…Indeed, it has been already been successful in helping to resolve a number of interesting new structures. Examples include a structure of FACT manipulating the nucleasome [19], multiple conformations of an ABC exporter [14], mTORC1 docked on the lysosome [24], the respiratory syncytial virus polymerase complex [9], a GPCR-G protein-β-arrestin megacomplex [20], and MERS-CoV/SARS-CoV with neutralizing antibodies [32]. An updated non-uniform refinement algorithm implementation, as described in this work, will be released in an upcoming version of cryoSPARC (www.cryosparc.com).…”

Section: Discussionmentioning

confidence: 99%

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Punjani

Zhang

Fleet

2019

Preprint

277

329

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Single particle cryo-EM is a powerful method for studying proteins and other biological macromolecules. Many of these molecules comprise regions with varying structural properties including disorder, flexibility, and partial occupancy. These traits make computational 3D reconstruction from 2D images challenging. Detergent micelles and lipid nanodiscs, used to keep membrane proteins in solution, are common examples of locally disordered structures that can negatively affect existing iterative refinement algorithms which assume rigidity (or spatial uniformity). We introduce a cross-validation approach to derive non-uniform refinement, an algorithm that automatically regularizes 3D density maps during iterative refinement to account for spatial variability, yielding dramatically improved resolution and 3D map quality. We find that in common iterative refinement methods, regularization using spatially uniform filtering operations can simultaneously over-and under-regularize local regions of a 3D map. In contrast, non-uniform refinement removes noise in disordered regions while retaining signal useful for aligning particle images. Our results include state-of-the-art resolution 3D reconstructions of multiple membrane proteins with molecular weight as low as 90kDa. These results demonstrate that higher resolutions and improved 3D density map quality can be achieved even for small membrane proteins, an important use case for single particle cryo-EM, both in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package and has already been used successfully in several notable structural studies.

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Section: Discussionmentioning

confidence: 99%

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Punjani

Zhang

Fleet

2019

Preprint

277

329

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“…Treating NPC1-null cells with Torin1 promoted clearance of autophagic material from the lysosomal lumen, as judged by both LC3B-LAMP2 double immunofluorescence ( Figure 4C-4D and S5A-S5B) and by direct immunoblotting of immunopurified lysosomal samples from both NPC1-KO HEK-293T and MEFs ( Figure 4E and S5C). Also, inhibiting mTORC1 signaling via shRNA-mediated knock down of LAMTOR5, which is essential for mTORC1 recruitment to and activation at the lysosome (but is not required for mTORC2-dependent signaling) (Anandapadamanaban et al, 2019;de Araujo et al, 2017;Bar-Peled et al, 2012;Rogala et al, 2019;Su et al, 2017), led to pronounced clearance of accumulated LC3 from the lumen of LAMP2positive NPC lysosomes ( Figure 4F-4G and S5D).…”

Section: Mtorc1 Inhibition Restores Integrity and Proteolytic Functiomentioning

confidence: 99%

NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

Davis

Shin

Lim

et al. 2020

Preprint

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Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function and neurodegeneration. The compositional and functional alterations in NPC lysosomes, and how aberrant cholesterol-mTORC1 signaling contributes to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion and enhanced membrane damage. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.

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“…This insertion is made possible because the space between the G-domains is widened compared to the structure of the free dimer [31]. Superimposition of the GTP-bound structure of RagC shows that in this conformation, there would be a clash between the RagC switch I and the tip of the claw, and the inter G-domain space would be narrower [32]. Modelling of different combinations of nucleotide loading in RagA and RagC revealed that the interactions observed are favored only in the active conformation.…”

Section: Mtorc1 Activation: Rag and Rheb Gtpasesmentioning

confidence: 99%

Structural Insights into TOR Signaling

Tafur

Kefauver

Loewith

2020

Genes

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The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.

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Structural basis for the docking of mTORC1 on the lysosomal surface

Cited by 141 publications

References 79 publications

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

Structural Insights into TOR Signaling

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