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1.
Sci Adv ; 9(37): eadd9084, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37703363

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.


Assuntos
Actinas , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina , Citoesqueleto de Actina , Lisossomos
2.
Elife ; 102021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33856341

RESUMO

Numerous proteins target lipid droplets (LDs) through amphipathic helices (AHs). It is generally assumed that AHs insert bulky hydrophobic residues in packing defects at the LD surface. However, this model does not explain the targeting of perilipins, the most abundant and specific amphipathic proteins of LDs, which are weakly hydrophobic. A striking example is Plin4, whose gigantic and repetitive AH lacks bulky hydrophobic residues. Using a range of complementary approaches, we show that Plin4 forms a remarkably immobile and stable protein layer at the surface of cellular or in vitro generated oil droplets, and decreases LD size. Plin4 AH stability on LDs is exquisitely sensitive to the nature and distribution of its polar residues. These results suggest that Plin4 forms stable arrangements of adjacent AHs via polar/electrostatic interactions, reminiscent of the organization of apolipoproteins in lipoprotein particles, thus pointing to a general mechanism of AH stabilization via lateral interactions.


Assuntos
Gotículas Lipídicas/metabolismo , Perilipina-4/química , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Estrutura Secundária de Proteína
3.
Adv Sci (Weinh) ; 8(17): e2101614, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34250755

RESUMO

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.


Assuntos
Aminoácidos/metabolismo , Neoplasias da Mama/metabolismo , Endocitose , Matriz Extracelular/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos
4.
Nat Commun ; 9(1): 1332, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29626194

RESUMO

How proteins are targeted to lipid droplets (LDs) and distinguish the LD surface from the surfaces of other organelles is poorly understood, but many contain predicted amphipathic helices (AHs) that are involved in targeting. We have focused on human perilipin 4 (Plin4), which contains an AH that is exceptional in terms of length and repetitiveness. Using model cellular systems, we show that AH length, hydrophobicity, and charge are important for AH targeting to LDs and that these properties can compensate for one another, albeit at a loss of targeting specificity. Using synthetic lipids, we show that purified Plin4 AH binds poorly to lipid bilayers but strongly interacts with pure triglycerides, acting as a coat and forming small oil droplets. Because Plin4 overexpression alleviates LD instability under conditions where their coverage by phospholipids is limiting, we propose that the Plin4 AH replaces the LD lipid monolayer, for example during LD growth.


Assuntos
Gotículas Lipídicas/metabolismo , Perilipina-4/química , Perilipina-4/metabolismo , Animais , Linhagem Celular , Drosophila , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Gotículas Lipídicas/química , Modelos Moleculares , Perilipina-4/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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