The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.

• Klíčová slova
• biochemistry, mTOR signaling, nutrient sensing,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aminokyseliny * metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• lyzozomy * metabolismus   MeSH
• monomerní proteiny vázající GTP * metabolismus   MeSH
• mTORC1 * metabolismus   MeSH
• myši MeSH
• signální transdukce * MeSH
• živiny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• aminokyseliny * MeSH
• monomerní proteiny vázající GTP * MeSH
• mTORC1 * MeSH
• RRAGA protein, human MeSH Prohlížeč
• RRAGC protein, human MeSH Prohlížeč

Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system.

• MeSH
• Drosophila melanogaster * metabolismus   MeSH
• mTORC1 metabolismus   MeSH
• multiproteinové komplexy metabolismus   MeSH
• S-adenosylmethionin MeSH
• savci metabolismus   MeSH
• TOR serin-threoninkinasy * metabolismus   MeSH
• živiny MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mTORC1 MeSH
• multiproteinové komplexy MeSH
• S-adenosylmethionin MeSH
• TOR serin-threoninkinasy * MeSH

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMP-activated protein kinase and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin-positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk, and organism longevity.

• Klíčová slova
• AMPK, C. elegans, aging, cell biology, longevity, lysosome, organelle interaction,
• MeSH
• intracelulární membrány metabolismus   MeSH
• lyzozomy * metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

• Klíčová slova
• SEGA, TSC, low grade glioma, sequencing,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• astrocytom etiologie   genetika   metabolismus   MeSH
• astrocyty účinky léků   metabolismus   MeSH
• butadieny farmakologie   MeSH
• dítě MeSH
• dospělí MeSH
• extracelulárním signálem regulované MAP kinasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• hamartin genetika   MeSH
• inhibitory enzymů farmakologie   MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• kojenec MeSH
• lidé MeSH
• MAP kinasový signální systém genetika   MeSH
• messenger RNA metabolismus   MeSH
• mikro RNA metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mTORC1 MeSH
• nádorové buňky kultivované MeSH
• nádory mozku komplikace   genetika   metabolismus   MeSH
• nitrily farmakologie   MeSH
• předškolní dítě MeSH
• sekvenční analýza RNA MeSH
• sekvenování transkriptomu MeSH
• stanovení celkové genové exprese MeSH
• tuberin genetika   MeSH
• tuberózní skleróza komplikace   genetika   MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• butadieny MeSH
• extracelulárním signálem regulované MAP kinasy MeSH
• hamartin MeSH
• inhibitory enzymů MeSH
• intracelulární signální peptidy a proteiny MeSH
• LAMTOR1 protein, human MeSH Prohlížeč
• LAMTOR2 protein, human MeSH Prohlížeč
• LAMTOR3 protein, human MeSH Prohlížeč
• LAMTOR4 protein, human MeSH Prohlížeč
• LAMTOR5 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• mikro RNA MeSH
• mTORC1 MeSH
• nitrily MeSH
• TSC1 protein, human MeSH Prohlížeč
• TSC2 protein, human MeSH Prohlížeč
• tuberin MeSH
• U 0126 MeSH Prohlížeč
• výměnné faktory guaninnukleotidů MeSH

Proteins of the mammalian target of rapamycin (mTOR) signaling axis are overexpressed or mutated in cancers. However, clinical inhibition of mTOR signaling as a therapeutic strategy in oncology shows rather limited progress. Nanoparticle-based mTOR targeted therapy proposes an attractive therapeutic option for various types of cancers. Along with the progress in the biomedical applications of nanoparticles, we start to realize the challenges and opportunities that lie ahead. Here, we critically analyze the current literature on the modulation of mTOR activity by nanoparticles, demonstrate the complexity of cellular responses to functionalized nanoparticles, and underline challenges lying in the identification of the molecular mechanisms of mTOR signaling affected by nanoparticles. We propose the idea that subcytotoxic doses of nanoparticles could be relevant for the induction of subcellular structural changes with possible involvement of mTORC1 signaling. The evaluation of the mechanisms and therapeutic effects of nanoparticle-based mTOR modulation will provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

• Klíčová slova
• lysosomes, mTOR, molecular targeting, nanoparticles, reactive oxygen species,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization and biodegradability. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH2), but not amino- or hydroxyl-functionalized silica particles, trigger cell death in hepatocellular carcinoma Huh7 cells. Importantly, biodegradability of nanoparticles plays a crucial role in regulation of essential cellular processes. Thus, biodegradable silica nanoparticles having the same shape, size and surface functionalization showed opposite cellular effects in comparison with similar polystyrene nanoparticles. At the molecular level, PS-NH2 obstruct and amino-functionalized silica nanoparticles (Si-NH2) activate the mTOR signalling in Huh7 and HepG2 cells. PS-NH2 induced time-dependent lysosomal destabilization associated with damage of the mitochondrial membrane. Solely in PS-NH2-treated cells, permeabilization of lysosomes preceded cell death. Contrary, Si-NH2 nanoparticles enhanced proliferation of HuH7 and HepG2 cells. Our findings demonstrate complex cellular responses to functionalized nanoparticles and suggest that nanoparticles can be used to control activation of mTOR signaling with subsequent influence on proliferation and viability of HuH7 cells. The data provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

• MeSH
• adsorpce MeSH
• aminy chemie   MeSH
• hepatocelulární karcinom metabolismus   patologie   MeSH
• konformace proteinů MeSH
• lyzozomy metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory jater metabolismus   patologie   MeSH
• nanočástice chemie   MeSH
• oxid křemičitý chemie   MeSH
• permeabilita MeSH
• polystyreny chemie   MeSH
• povrchové vlastnosti MeSH
• proliferace buněk MeSH
• ribonukleasy metabolismus   MeSH
• sérový albumin hovězí metabolismus   MeSH
• signální transdukce * MeSH
• skot MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• oxid křemičitý MeSH
• polystyreny MeSH
• ribonukleasy MeSH
• sérový albumin hovězí MeSH
• TOR serin-threoninkinasy MeSH

• Klíčová slova
• LC3, autolysosome, autophagosome, chaperone-mediated autophagy, flux, lysosome, macroautophagy, phagophore, stress, vacuole,
• MeSH
• autofagie * fyziologie   MeSH
• biotest metody   normy   MeSH
• lidé MeSH
• počítačová simulace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• směrnice MeSH