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1.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
2.
Nat Commun ; 11(1): 3306, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620754

RESUMO

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
Proc Natl Acad Sci U S A ; 117(13): 7482-7493, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32170020

RESUMO

Plants balance their competing requirements for growth and stress tolerance via a sophisticated regulatory circuitry that controls responses to the external environments. We have identified a plant-specific gene, COST1 (constitutively stressed 1), that is required for normal plant growth but negatively regulates drought resistance by influencing the autophagy pathway. An Arabidopsis thaliana cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes, while overexpression of COST1 confers drought hypersensitivity and reduced autophagy. The COST1 protein is degraded upon plant dehydration, and this degradation is reduced upon treatment with inhibitors of the 26S proteasome or autophagy pathways. The drought resistance of a cost1 mutant is dependent on an active autophagy pathway, but independent of other known drought signaling pathways, indicating that COST1 acts through regulation of autophagy. In addition, COST1 colocalizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and affects the level of ATG8e protein through physical interaction with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. Under drought, COST1 is degraded, enabling activation of autophagy and suppression of growth to enhance drought tolerance. Our research places COST1 as an important regulator controlling the balance between growth and stress responses via the direct regulation of autophagy.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Estresse Fisiológico/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagossomos/metabolismo , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Secas , Genes de Plantas , Transdução de Sinais , Estresse Fisiológico/genética
4.
Nat Cell Biol ; 22(2): 159-166, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029894

RESUMO

Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia/genética , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Citoplasma/metabolismo , Glucose/farmacologia , Complexos Multiproteicos/metabolismo , Nitrogênio/farmacologia , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia
5.
Mol Cell ; 77(6): 1163-1175.e9, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31995729

RESUMO

Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.


Assuntos
Aminopeptidases/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Aminopeptidases/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Citoplasma/metabolismo , Mutação , Ligação Proteica , Transporte Proteico , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Proteínas de Transporte Vesicular/genética
6.
PLoS One ; 15(1): e0227603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940411

RESUMO

In-vitro studies with different Fanconi anemia (FA) cell lines and FANC gene silenced cell lines indicating involvement of mitochondria function in pathogenesis of FA have been reported. However, in-vivo studies have not been studied so far to understand the role of mitochondrial markers in pathogenesis of FA. We have carried out a systematic set of biomarker studies for elucidating involvement of mitochondrial dysfunction in disease pathogenesis for Indian FA patients. We report changes in the mtDNA number in 59% of FA patients studied, a high frequency of mtDNA variations (37.5% of non-synonymous variations and 62.5% synonymous variations) and downregulation of mtDNA complex-I and complex-III encoding genes of OXPHOS (p<0.05) as strong biomarkers for impairment of mitochondrial functions in FA. Deregulation of expression of mitophagy genes (ATG; p>0.05, Beclin-1; p>0.05, and MAP1-LC3, p<0.05) has also been observed, suggesting inability of FA cells to clear off impaired mitochondria. We hypothesize that accumulation of such impaired mitochondria in FA cells therefore may be the principal cause for bone marrow failure (BMF) and a plausible effect of inefficient clearance of impaired mitochondria in FA.


Assuntos
DNA Mitocondrial/genética , Anemia de Fanconi/genética , Adolescente , Adulto , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Criança , DNA Mitocondrial/metabolismo , Anemia de Fanconi/metabolismo , Feminino , Variação Genética/genética , Humanos , Índia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Espécies Reativas de Oxigênio/metabolismo
7.
J Mol Biol ; 432(1): 80-103, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31310766

RESUMO

Selective autophagy relies on soluble or membrane-bound cargo receptors that recognize cargo and bring about autophagosome formation at the cargo. The cargo-bound receptors interact with lipidated ATG8 family proteins anchored in the membrane at the concave side of the forming autophagosome. The interaction is mediated by 15- to 20-amino-acid-long sequence motifs called LC3-interacting region (LIR) motifs that bind to the LIR docking site (LDS) of ATG8 proteins. In this review, we focus on LIR-ATG8 interactions and the soluble mammalian selective autophagy receptors. We discuss the roles of ATG8 family proteins as membrane scaffolds in autophagy and the LIR-LDS interaction and how specificity for binding to GABARAP or LC3 subfamily proteins is achieved. We also discuss atypical LIR-LDS interactions and a novel LIR-independent interaction. Recently, it has become clear that several of the soluble cargo receptors are able to recruit components of the core autophagy apparatus to aid in assembling autophagosome formation at the site of cargo sequestration. A model on phagophore recruitment and expansion on a selective autophagy receptor-coated cargo incorporating the latest findings is presented.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Animais , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/química , Família da Proteína 8 Relacionada à Autofagia/análise , Humanos , Macroautofagia , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas
8.
Autophagy ; 16(1): 123-139, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30909785

RESUMO

Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In Arabidopsis thaliana, the core machinery of autophagy is well defined, but its transcriptional regulation is largely unknown. The ATG8 (autophagy-related 8) protein plays central roles in decorating autophagosomes and binding to specific cargo receptors to recruit cargo to autophagosomes. We propose that the transcriptional control of ATG8 genes is important during the formation of autophagosomes and therefore contributes to survival during stress. Here, we describe a yeast one-hybrid (Y1H) screen for transcription factors (TFs) that regulate ATG8 gene expression in Arabidopsis, using the promoters of 4 ATG8 genes. We identified a total of 225 TFs from 35 families that bind these promoters. The TF-ATG8 promoter interactions revealed a wide array of diverse TF families for different promoters, as well as enrichment for families of TFs that bound to specific fragments. These TFs are not only involved in plant developmental processes but also in the response to environmental stresses. TGA9 (TGACG (TGA) motif-binding protein 9)/AT1G08320 was confirmed as a positive regulator of autophagy. TGA9 overexpression activated autophagy under both control and stress conditions and transcriptionally up-regulated expression of ATG8B, ATG8E and additional ATG genes via binding to their promoters. Our results provide a comprehensive resource of TFs that regulate ATG8 gene expression and lay a foundation for understanding the transcriptional regulation of plant autophagy.Abbreviations: ABRC: Arabidopsis biological resource center; AP2-EREBP: APETALA2/Ethylene-responsive element binding protein; ARF: auxin response factor; ATF4: activating transcription factor 4; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; DAP-seq: DNA affinity purification sequencing; FOXO: forkhead box O; GFP: green fluorescent protein; GO: gene ontologies; HB: homeobox; LD: long-day; LUC: firefly luciferase; MAP1LC3: microtubule associated protein 1 light chain 3; MDC: monodansylcadaverine; 3-MA: 3-methyladenine; OE: overexpressing; PCD: programmed cell death; qPCR: quantitative polymerase chain reaction; REN: renilla luciferase; RT: room temperature; SD: standard deviation; TF: transcription factor; TFEB: transcription factor EB; TGA: TGACG motif; TOR: target of rapamycin; TSS: transcription start site; WT: wild-type; Y1H: yeast one-hybrid.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Autofagossomos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Proteínas de Plantas , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/fisiologia
9.
Autophagy ; 16(2): 239-255, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982432

RESUMO

Autophagy is a conserved adaptive cellular pathway essential to maintain a variety of physiological functions. Core components of this machinery are the six human Atg8 orthologs that initiate formation of appropriate protein complexes. While these proteins are routinely used as indicators of autophagic flux, it is presently not possible to discern their individual biological functions due to our inability to predict specific binding partners. In our attempts towards determining downstream effector functions, we developed a computational pipeline to define structural determinants of human Atg8 family members that dictate functional diversity. We found a clear evolutionary separation between human LC3 and GABARAP subfamilies and also defined a novel sequence motif responsible for their specificity. By analyzing known protein structures, we observed that functional modules or microclusters reveal a pattern of intramolecular network, including distinct hydrogen bonding of key residues (F52/Y49; a subset of HP2) that may directly modulate their interaction preferences. Multiple molecular dynamics simulations were performed to characterize how these proteins interact with a common protein binding partner, PLEKHM1. Our analysis showed remarkable differences in binding modes via intrinsic protein dynamics, with PLEKHM1-bound GABARAP complexes showing less fluctuations and higher number of contacts. We further mapped 373 genomic variations and demonstrated that distinct cancer-related mutations are likely to lead to significant structural changes. Our findings present a quantitative framework to establish factors underlying exquisite specificity of human Atg8 proteins, and thus facilitate the design of precise modulators.Abbreviations: Atg: autophagy-related; ECs: evolutionary constraints; GABARAP: GABA type A receptor-associated protein; HsAtg8: human Atg8; HP: hydrophobic pocket; KBTBD6: kelch repeat and BTB domain containing 6; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MD: molecular dynamics; HIV-1 Nef: human immunodeficiency virus type 1 negative regulatory factor; PLEKHM1: pleckstrin homology and RUN domain containing M1; RMSD: root mean square deviation; SQSTM1/p62: sequestosome 1; WDFY3/ALFY: WD repeat and FYVE domain containing 3.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Sítios de Ligação , Evolução Molecular , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Mutação/genética , Neoplasias/genética , Ligação Proteica , Relação Estrutura-Atividade
10.
Autophagy ; 16(2): 256-270, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-30990354

RESUMO

Short linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/química , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lisina/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
11.
J Biol Chem ; 295(5): 1240-1260, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31857374

RESUMO

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.


Assuntos
Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutação , Quinases Relacionadas a NIMA/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/genética , Espectrometria de Massas em Tandem , Treonina/metabolismo
12.
BMB Rep ; 52(12): 700-705, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31722778

RESUMO

The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways. [BMB Reports 2019; 52(12): 700-705].


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/genética , Motivos de Aminoácidos/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/antagonistas & inibidores , Proteínas de Bactérias/genética , Células Cultivadas , Fibroblastos , Células HEK293 , Humanos , Legionella pneumophila/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética
13.
Int J Mol Sci ; 20(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744172

RESUMO

Although studies have shown the concomitant occurrence of autophagic and programmed cell death (PCD) in plants, the relationship between autophagy and PCD and the factors determining this relationship remain unclear. In this study, seedlings of the wheat cultivar Jimai 22 were used to examine the occurrence of autophagy and PCD during polyethylene glycol (PEG)-8000-induced drought stress. Autophagy and PCD occurred sequentially, with autophagy at a relatively early stage and PCD at a much later stage. These findings suggest that the duration of drought stress determines the occurrence of PCD following autophagy. Furthermore, the addition of 3-methyladenine (3-MA, an autophagy inhibitor) and the knockdown of autophagy-related gene 6 (ATG6) accelerated PEG-8000-induced PCD, respectively, suggesting that inhibition of autophagy also results in PCD under drought stress. Overall, these findings confirm that wheat seedlings undergo autophagic survival under mild drought stress, with subsequent PCD only under severe drought.


Assuntos
Apoptose , Autofagia , Secas , Triticum/crescimento & desenvolvimento , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Polietilenoglicóis/toxicidade , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Triticum/metabolismo
14.
Sci Rep ; 9(1): 14828, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31616012

RESUMO

Cellular heat stress can cause damage, and significant changes, to a variety of cellular structures. When exposed to chronically high temperatures, yeast cells invaginate vacuolar membranes. In this study, we found that the expression of Atg8, an essential autophagy factor, is induced after chronic heat stress. In addition, without Atg8, vacuolar invaginations are induced conspicuously, beginning earlier and invaginating vacuoles more frequently after heat stress. Our results indicate that Atg8's invagination-suppressing functions do not require Atg8 lipidation, in contrast with autophagy, which requires Atg8 lipidation. Genetic analyses of vps24 and vps23 further suggest that full ESCRT machinery is necessary to form vacuolar invaginations irrespective of Atg8. In contrast, through a combined mutation with the vacuole BAR domain protein Ivy1, vacuoles show constitutively enhanced invaginated structures. Finally, we found that the atg8Δivy1Δ mutant is sensitive against agents targeting functions of the vacuole and/or plasma membrane (cell wall). Collectively, our findings revealed that Atg8 maintains vacuolar membrane homeostasis in an autophagy-independent function by coordinating with other cellular factors.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Resposta ao Choque Térmico , Mutação , Proteínas de Saccharomyces cerevisiae/genética
15.
EMBO J ; 38(22): e101994, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31625181

RESUMO

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Lisossomos/metabolismo , Proteínas Qa-SNARE/metabolismo , Sintaxina 16/metabolismo , Motivos de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/genética , Células HEK293 , Células HeLa , Humanos , Redes e Vias Metabólicas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , RNA Interferente Pequeno/genética , Sintaxina 16/antagonistas & inibidores , Sintaxina 16/genética
16.
Int J Mol Sci ; 20(19)2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31597279

RESUMO

Autophagy, a conserved pathway in eukaryotes, degrades and recycles cellular components, thus playing an important role in nitrogen (N) remobilization. N plays an important role in the growth and development of plants, which also affects plant yield and quality. In this research, it was found that the transcriptional level of a core autophagy gene of rice (Oryza sativa), OsATG8c, was increased during N starvation conditions. It was found that the overexpression of OsATG8c significantly enhanced the activity of autophagy and that the number of autophagosomes, dwarfed the plant height and increased the effective tillers' number and yield. The nitrogen uptake efficiency (NUpE) and nitrogen use efficiency (NUE) significantly increased in the transgenic rice under both optimal and suboptimal N conditions. Based on our results, OsATG8c is considered to be a good candidate gene for increasing NUE, especially under suboptimal field conditions.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia/genética , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Oryza/genética , Oryza/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Desenvolvimento Vegetal , Plantas Geneticamente Modificadas , Transporte Proteico
17.
Mol Plant Pathol ; 20(9): 1211-1216, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397085

RESUMO

Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to 'non-self' such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy-virus interplay and its consequences for host immunity and viral pathogenesis in plants.


Assuntos
Autofagia/fisiologia , Vírus/patogenicidade , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Vírus/imunologia
18.
Nat Commun ; 10(1): 3600, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399562

RESUMO

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12-Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3's catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3's E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.


Assuntos
Regulação Alostérica/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Domínio Catalítico , Cristalografia por Raios X , Ligases , Modelos Moleculares , Mutação , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/metabolismo
19.
J Biol Chem ; 294(38): 14033-14042, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31362979

RESUMO

Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Autofagia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mitofagia , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia
20.
Cells ; 8(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466295

RESUMO

Autophagy is a highly conserved catabolic process in eukaryotic cells by which waste cellular components are recycled to maintain growth in both favorable and stress conditions. Autophagy has been linked to lipid metabolism in microalgae; however, the mechanism underlying this interaction remains unclear. In this study, transgenic Chlamydomonas reinhardtii cells that stably express the red fluorescent protein (mCherry) tagged-ATG8 as an autophagy marker were established. By using this tool, we were able to follow the autophagy process in live microalgal cells under various conditions. Live-cell and transmission electron microscopy (TEM) imaging revealed physical contacts between lipid droplets and autophagic structures during the early stage of nitrogen starvation, while fusion of these two organelles was observed in prolonged nutritional deficiency, suggesting that an autophagy-related pathway might be involved in lipid droplet turnover in this alga. Our results thus shed light on the interplay between autophagy and lipid metabolism in C. reinhardtii, and this autophagy marker would be a valuable asset for further investigations on autophagic processes in microalgae.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Chlamydomonas reinhardtii/genética , Gotículas Lipídicas/metabolismo , Autofagossomos/ultraestrutura , Autofagia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Cloroquina/efeitos adversos , Gotículas Lipídicas/ultraestrutura , Metabolismo dos Lipídeos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Transmissão , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/ultraestrutura
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