Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.422
Filtrar
1.
Biochem Pharmacol ; 208: 115406, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36596415

RESUMO

SAP97 is a member of the MAGUK family of proteins, but unlike other MAGUK proteins that are selectively expressed in the CNS, SAP97 is also expressed in peripheral organs, like the heart and kidneys. SAP97 has several protein binding cassettes, and this review will describe their involvement in creating SAP97-anchored multiprotein networks. SAP97-anchored networks localized at the inner leaflet of the cell membrane play a major role in trafficking and targeting of membrane G protein-coupled receptors (GPCR), channels, and structural proteins. SAP97 plays a major role in compartmentalizing voltage gated sodium and potassium channels to specific cellular compartments of heart cells. SAP97 undergoes extensive alternative splicing. These splice variants give rise to different SAP97 isoforms that alter its cellular localization, networking, signaling and trafficking effects. Regarding GPCR, SAP97 binds to the ß1-adrenergic receptor and recruits AKAP5/PKA and PDE4D8 to create a multiprotein complex that regulates trafficking and signaling of cardiac ß1-AR. In the kidneys, SAP97 anchored networks played a role in trafficking of aquaporin-2 water channels. Cardiac specific ablation of SAP97 (SAP97-cKO) resulted in cardiac hypertrophy and failure in aging mice. Similarly, instituting transverse aortic constriction (TAC) in young SAP97 c-KO mice exacerbated TAC-induced cardiac remodeling and dysfunction. These findings highlight a critical role for SAP97 in the pathophysiology of a number of cardiac and renal diseases, suggesting that SAP97 is a relevant target for drug discovery.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana , Camundongos , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo
2.
Mol Cell ; 83(1): 74-89.e9, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36528027

RESUMO

The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.


Assuntos
Complexos Multiproteicos , Serina-Treonina Quinases TOR , Animais , Camundongos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais
3.
Proc Natl Acad Sci U S A ; 120(1): e2212330120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577058

RESUMO

Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. Here we report that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. We find that in the ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, our data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context.


Assuntos
Proteínas de Drosophila , Drosophila , Complexos Multiproteicos , Proteína Fosfatase 2 , Animais , Feminino , Humanos , Anticorpos , Drosophila/metabolismo , Proteínas Ativadoras de GTPase , Células HeLa , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Drosophila/metabolismo
4.
J Immunol ; 209(12): 2287-2291, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36469844

RESUMO

The mechanistic target of rapamycin is an essential regulator of T cell metabolism and differentiation. In this study, we demonstrate that serum- and glucocorticoid-regulated kinase 1 (SGK1), a downstream node of mechanistic target of rapamycin complex 2 signaling, represses memory CD8+ T cell differentiation. During acute infections, murine SGK1-deficient CD8+ T cells adopt an early memory precursor phenotype leading to more long-lived memory T cells. Thus, SGK1-deficient CD8+ T cells demonstrate an enhanced recall capacity in response to reinfection and can readily reject tumors. Mechanistically, activation of SGK1-deficient CD8+ T cells results in decreased Foxo1 phosphorylation and increased nuclear translocation of Foxo1 to promote early memory development. Overall, SGK1 might prove to be a powerful target for enhancing the efficacy of vaccines and tumor immunotherapy.


Assuntos
Glucocorticoides , Complexos Multiproteicos , Camundongos , Animais , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Glucocorticoides/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células T de Memória , Linfócitos T CD8-Positivos , Sirolimo , Diferenciação Celular , Memória Imunológica/genética
5.
Yakugaku Zasshi ; 142(11): 1191-1199, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-36328449

RESUMO

The role of ß-estradiol (E2) in lipoprotein metabolism in mammary tumors remains unknown. Therefore the effect of E2 on secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells was examined. The E2-treated FM3A cells increased active LPL secretion in a time- and dose-dependent manner. The activity of mitogen-activated protein kinase (MAPK) was elevated in the tumor cells treated with E2, and E2-stimulated secretion of LPL was suppressed by the MAPK kinase 1/2 inhibitor PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor FR180204, p38 MAPK inhibitor SB202190, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In addition, the effect of E2 on active LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but not by the mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed the secretion of LPL by E2. Stimulatory secretion of LPL by E2 from the tumor cells is closely associated with activation of mTORC2 rather than mTORC1, possibly via the MAPK cascade.


Assuntos
Complexos Multiproteicos , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Estradiol/farmacologia , Lipoproteínas , Mamíferos/genética , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno , Sirolimo/farmacologia , Neoplasias da Mama/metabolismo
6.
Biomolecules ; 12(11)2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36358936

RESUMO

Autophagy-dependent cellular survival is tightly regulated by both kinases and phosphatases. While mTORC1 inhibits autophagy by phosphorylating ULK1, PP2A is able to remove this phosphate group from ULK1 and promotes the key inducer of autophagosome formation. However, ULK1 inhibits mTORC1, mTORC1 is able to down-regulate PP2A. In addition, the active ULK1 promotes PP2A via phosphorylation. We claim that these double-negative (mTORC1 -| PP2A -| mTORC1, mTORC1 -| ULK1 -| mTORC1) and positive (ULK1 -> PP2A -> ULK1) feedback loops are all necessary for the robust, irreversible decision making process between the autophagy and non-autophagy states. We approach our scientific analysis from a systems biological perspective by applying both theoretical and molecular biological techniques. For molecular biological experiments, HEK293T cell line is used, meanwhile the dynamical features of the regulatory network are described by mathematical modelling. In our study, we explore the dynamical characteristic of mTORC1-ULK1-PP2A regulatory triangle in detail supposing that the positive feedback loops are essential to manage a robust cellular answer upon various cellular stress events (such as mTORC1 inhibition, starvation, PP2A inhibition or ULK1 silencing). We confirm that active ULK1 can up-regulate PP2A when mTORC1 is inactivated. By using theoretical analysis, we explain the importance of cellular PP2A level in stress response mechanism. We proved both experimentally and theoretically that PP2A down-regulation (via addition of okadaic acid) might generate a periodic repeat of autophagy induction. Understanding how the regulation of the cell survival occurs with the precise molecular balance of ULK1-mTORC1-PP2A in autophagy, is highly relevant in several cellular stress-related diseases (such as neurodegenerative diseases or diabetes) and might help to promote advanced therapies in the near future, too.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Serina-Treonina Quinases TOR , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células HEK293 , Fosforilação
7.
J Proteome Res ; 21(12): 2893-2904, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36315652

RESUMO

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Glioblastoma/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Complexos Multiproteicos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias Encefálicas/metabolismo , Dano ao DNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo
8.
Mol Nutr Food Res ; 66(23): e2200186, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36189894

RESUMO

SCOPE: Mechanistic target of rapamycin (mTOR) serves as a central signaling node in the coordination of cell growth and metabolism, and it functions via two distinct complexes, namely, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 plays a crucial role in sensing amino acids, whereas mTORC2 involves in sensing growth factors. However, it remains largely unclear whether mTORC2 can sense amino acids and the mechanism by which amino acids regulate mTORC2 has not been studied. METHODS AND RESULTS: After treating cells with indicated concentration of amino acids for different time, it is found that the mTORC2 activation is significantly increased in response to amino acids stimulation, especially cystine. Particularly, knockdown solute carrier family 7 member 11 (SLC7A11) by siRNA shows that SLC7A11-mediated cystine uptake is responsible for activating mTORC2. Mechanistically, the study finds that p38 is activated in response to cystine stimulation, and co-immunoprecipitation (Co-IP) experiments suggest that p38 regulates the assembly of components within mTORC2 by mediating the phosphorylation of the mTORC2 subunit mitogen-activated protein kinase-interacting protein 1 (Sin1) in a cystine-dependent manner. Finally, combined with inducers and inhibitors of ferroptosis and cell viability assay, the study observes that cystine-mediated regulation of the p38-Sin1-mTOR-AKT pathway induces resistance to ferroptosis. CONCLUSION: These results indicate that cystine-induced activation of the p38-Sin1-mTORC2-AKT pathway suppresses ferroptosis.


Assuntos
Ferroptose , Neoplasias , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Cistina/farmacologia , Cistina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
9.
Mol Cell ; 82(22): 4202-4217.e5, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36302374

RESUMO

Condensins are evolutionarily conserved molecular motors that translocate along DNA and form loops. To address how DNA topology affects condensin translocation, we applied auxin-inducible degradation of topoisomerases I and II and analyzed the binding and function of an interphase condensin that mediates X chromosome dosage compensation in C. elegans. TOP-2 depletion reduced long-range spreading of condensin-DC (dosage compensation) from its recruitment sites and shortened 3D DNA contacts measured by Hi-C. TOP-1 depletion did not affect long-range spreading but resulted in condensin-DC accumulation within expressed gene bodies. Both TOP-1 and TOP-2 depletion resulted in X chromosome derepression, indicating that condensin-DC translocation at both scales is required for its function. Together, the distinct effects of TOP-1 and TOP-2 suggest two distinct modes of condensin-DC association with chromatin: long-range DNA loop extrusion that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.


Assuntos
Adenosina Trifosfatases , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Adenosina Trifosfatases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismo , Cromossomos/metabolismo
10.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36232288

RESUMO

Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and shortening of a poly(A) tail are regulated by large multiprotein complexes. However, the molecular mechanisms of these protein machineries still remain unclear. Recent studies regarding the structural and biochemical characteristics of those protein complexes have shed light on the potential mechanisms of polyadenylation and deadenylation. This review summarizes the recent structural studies on pre-mRNA 3'-end processing complexes that initiate the polyadenylation and discusses the similarities and differences between yeast and human machineries. Specifically, we highlight recent biochemical efforts in the reconstitution of the active human canonical pre-mRNA 3'-end processing systems, as well as the roles of RBBP6/Mpe1 in activating the entire machinery. We also describe how poly(A) tails are removed by the PAN2-PAN3 and CCR4-NOT deadenylation complexes and discuss the emerging role of the cytoplasmic poly(A)-binding protein (PABPC) in promoting deadenylation. Together, these recent discoveries show that the dynamic features of these machineries play important roles in regulating polyadenylation and deadenylation.


Assuntos
Poliadenilação , Proteínas de Saccharomyces cerevisiae , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/genética , Humanos , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
11.
Nat Cell Biol ; 24(9): 1407-1421, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36097071

RESUMO

Mechanistic 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.


Assuntos
Complexos Multiproteicos , Transdução de Sinais , Animais , Encéfalo/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
12.
Nucleic Acids Res ; 50(18): 10680-10694, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36169232

RESUMO

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3'UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3'UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3'UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Complexos Multiproteicos/metabolismo , Regiões 3' não Traduzidas , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Humanos , Interferon gama/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética
13.
Front Cell Infect Microbiol ; 12: 979996, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36171757

RESUMO

The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.


Assuntos
Dengue , Complexos Multiproteicos , Apoptose , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral
14.
Methods Mol Biol ; 2548: 129-144, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36151496

RESUMO

Multiprotein complexes are important machineries that organize a large number of different proteins into functional units. Studying protein-protein interactions in the complexes, rather than individual proteins, is a fundamental step to gaining functional insights into a biological process. Here, we present the sequential affinity purification and coimmunoprecipitation system that was applied to enable the efficient purification of all the proteins that compose the Lpt system complex in Escherichia coli and their identification by western blotting and mass spectrometry (MS).


Assuntos
Escherichia coli , Proteínas , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoprecipitação , Complexos Multiproteicos/metabolismo , Proteínas/química
15.
Proc Natl Acad Sci U S A ; 119(37): e2123451119, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36067301

RESUMO

Filaggrin (FLG), an essential structural protein for skin barrier function, is down-regulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the -343/+25 of the FLG promoter was necessary for TNFα + IFNγ-induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ-induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene-induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.


Assuntos
Proteínas Filagrinas , Histona Desacetilase 1 , Queratinócitos , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Animais , Doença Crônica , Dermatite/genética , Dermatite/metabolismo , Regulação para Baixo , Proteínas Filagrinas/genética , Proteínas Filagrinas/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Interferon gama/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
16.
PLoS Biol ; 20(9): e3001737, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36099296

RESUMO

The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here, we show that vimentin, a cytoskeletal intermediate filament protein that we have known to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. This vimentin-mediated regulation is manifested at all levels of mTOR downstream target activation and protein synthesis. We found that vimentin maintains normal cell size by supporting mTORC1 translocation and activation by regulating the activity of amino acid sensing Rag GTPase. We also show that vimentin inhibits the autophagic flux in the absence of growth factors and/or critical nutrients, demonstrating growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings establish that vimentin couples cell size and autophagy through modulating Rag GTPase activity of the mTORC1 signaling pathway.


Assuntos
Filamentos Intermediários , Complexos Multiproteicos , Animais , Autofagia/fisiologia , Tamanho Celular , GTP Fosfo-Hidrolases/metabolismo , Filamentos Intermediários/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Vimentina/metabolismo
17.
Elife ; 112022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35983835

RESUMO

In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Although the mechanism of action and regulation of condensin I have been studied extensively, our corresponding knowledge of condensin II remains very limited. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We find that one of two HEAT subunits, CAP-D3, plays a crucial role in condensin II-mediated assembly of chromosome axes, whereas the other HEAT subunit, CAP-G2, has a very strong negative impact on this process. The structural maintenance of chromosomes ATPase and the basic amino acid clusters of the kleisin subunit CAP-H2 are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications for the evolution of condensin II.


Assuntos
Adenosina Trifosfatases , Complexos Multiproteicos , Adenosina Trifosfatases/metabolismo , Animais , Cromossomos/metabolismo , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo
18.
Diabetes ; 71(11): 2297-2312, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35983955

RESUMO

The innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that mice with diet-induced obesity (DIO) but not lean mice bearing a whole-body "TBK1-resistant" Mtor S2159A knock-in allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knock-in in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boost mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.


Assuntos
Hiperglicemia , Resistência à Insulina , Camundongos , Animais , Resistência à Insulina/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Sirolimo/farmacologia , Insulina/metabolismo , Obesidade/genética , Camundongos Obesos , Hiperglicemia/genética , Glucose , Proteínas Serina-Treonina Quinases/genética
19.
Proc Natl Acad Sci U S A ; 119(36): e2205669119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037331

RESUMO

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.


Assuntos
Aminoacil-tRNA Sintetases , Aminoacil-tRNA Sintetases/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Ribossomos/metabolismo
20.
Nature ; 609(7926): 408-415, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35831509

RESUMO

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .


Assuntos
Microscopia Crioeletrônica , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos , Proteína Fosfatase 1 , Proteínas ras , Motivos de Aminoácidos , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação de Sentido Incorreto , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/ultraestrutura , Estabilidade Proteica , Quinases raf , Proteínas ras/química , Proteínas ras/metabolismo , Proteínas ras/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...