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1.
FEBS Lett ; 595(17): 2197-2207, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339521

RESUMO

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.


Assuntos
Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/citologia , Vacúolos/metabolismo , Autofagossomos/química , Autofagossomos/metabolismo , Autofagia , Congelamento , Membranas Intracelulares/química , Lipídeos de Membrana/química , Microscopia Eletrônica , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Vacúolos/química
2.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360890

RESUMO

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Proteoma , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Filogenia , Processamento de Proteína Pós-Traducional , Proteômica/métodos
3.
Commun Biol ; 4(1): 828, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211117

RESUMO

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Canais de Translocação SEC/metabolismo , Animais , Retículo Endoplasmático/efeitos dos fármacos , Glicoconjugados/farmacologia , Células HeLa , Humanos , Immunoblotting , Membranas Intracelulares/efeitos dos fármacos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Canais de Translocação SEC/genética , Partícula de Reconhecimento de Sinal/metabolismo
4.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208277

RESUMO

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteoma/análise , Proteoma/metabolismo , RNA Mensageiro/genética
5.
Science ; 373(6554): 586-590, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326243

RESUMO

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana PpPIEZO1 and PpPIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both PpPIEZO1 and PpPIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Bryopsida/metabolismo , Canais Iônicos/metabolismo , Proteínas de Plantas/metabolismo , Vacúolos/ultraestrutura , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Bryopsida/crescimento & desenvolvimento , Bryopsida/ultraestrutura , Cálcio/metabolismo , Sinalização do Cálcio , Citoplasma/metabolismo , Membranas Intracelulares/metabolismo , Canais Iônicos/genética , Proteínas de Plantas/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Tubo Polínico/ultraestrutura , Vacúolos/metabolismo
6.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299309

RESUMO

Rab GTPases are major coordinators of intracellular membrane trafficking, including vesicle transport, membrane fission, tethering, docking, and fusion events. Rab GTPases are roughly divided into two groups: conventional "small" Rab GTPases and atypical "large" Rab GTPases that have been recently reported. Some members of large Rab GTPases in mammals include Rab44, Rab45/RASEF, and Rab46. The genes of these large Rab GTPases commonly encode an amino-terminal EF-hand domain, coiled-coil domain, and the carboxyl-terminal Rab GTPase domain. A common feature of large Rab GTPases is that they express several isoforms in cells. For instance, Rab44's two isoforms have similar functions, but exhibit differential localization. The long form of Rab45 (Rab45-L) is abundantly distributed in epithelial cells. The short form of Rab45 (Rab45-S) is predominantly present in the testes. Both Rab46 (CRACR2A-L) and the short isoform lacking the Rab domain (CRACR2A-S) are expressed in T cells, whereas Rab46 is only distributed in endothelial cells. Although evidence regarding the function of large Rab GTPases has been accumulating recently, there are only a limited number of studies. Here, we report the recent findings on the large Rab GTPase family concerning their function in membrane trafficking, cell differentiation, related diseases, and knockout mouse phenotypes.


Assuntos
Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/metabolismo , Masculino , Mastócitos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Fenótipo , Domínios Proteicos , Linfócitos T/metabolismo , Proteínas rab de Ligação ao GTP/genética
7.
Adv Protein Chem Struct Biol ; 126: 307-343, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34090618

RESUMO

The maintenance of cellular homeostasis involves the participation of multiple organelles, such as the endoplasmic reticulum (ER) and mitochondria. Specifically, ER plays a key role in calcium (Ca2+) storage, lipid synthesis, protein folding, and assembly, while mitochondria are the "energy factories" and provide energy to drive intracellular processes. Hence, alteration in ER or mitochondrial homeostasis has detrimental effects on cell survival, being linked to the triggering of apoptosis, a programmed form of cell death. Besides, ER stress conditions affect mitochondria functionality and vice-versa, as ER and mitochondria communicate via mitochondria-associated ER membranes (MAMs) to carry out a number of fundamental cellular functions. It is not surprising, thus, that also MAMs perturbations are involved in the regulation of apoptosis. This chapter intends to accurately discuss the involvement of MAMs in apoptosis, highlighting their crucial role in controlling this delicate cellular process.


Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Animais , Humanos
8.
Methods Mol Biol ; 2276: 215-225, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060044

RESUMO

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.


Assuntos
Membranas Intracelulares/patologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/patologia , Morte Celular/fisiologia , Células Cultivadas , Citocromos c/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia
9.
Nat Commun ; 12(1): 3475, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108457

RESUMO

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.


Assuntos
Membranas Intracelulares/metabolismo , Tilacoides/metabolismo , Proteínas de Bactérias/metabolismo , Membranas Intracelulares/ultraestrutura , Luz , Microscopia Eletrônica , Modelos Biológicos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Multimerização Proteica , Proteômica , Synechococcus/crescimento & desenvolvimento , Synechococcus/metabolismo , Synechococcus/ultraestrutura , Tilacoides/ultraestrutura
10.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064125

RESUMO

To initiate infection, a virus enters a host cell typically via receptor-dependent endocytosis. It then penetrates a subcellular membrane, reaching a destination that supports transcription, translation, and replication of the viral genome. These steps lead to assembly and morphogenesis of the new viral progeny. The mature virus finally exits the host cell to begin the next infection cycle. Strikingly, viruses hijack host molecular chaperones to accomplish these distinct entry steps. Here we highlight how DNA viruses, including polyomavirus and the human papillomavirus, exploit soluble and membrane-associated chaperones to enter a cell, penetrating and escaping an intracellular membrane en route for infection. We also describe the mechanism by which RNA viruses-including flavivirus and coronavirus-co-opt cytosolic and organelle-selective chaperones to promote viral endocytosis, protein biosynthesis, replication, and assembly. These examples underscore the importance of host chaperones during virus infection, potentially revealing novel antiviral strategies to combat virus-induced diseases.


Assuntos
Vírus de DNA/fisiologia , Chaperonas Moleculares/metabolismo , Vírus de RNA/fisiologia , Citosol/metabolismo , Vírus de DNA/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/metabolismo , Vírus de RNA/metabolismo , Internalização do Vírus , Replicação Viral
11.
Artigo em Inglês | MEDLINE | ID: mdl-33945875

RESUMO

The biogenesis of peroxisomes in relation to the trafficking of proteins to peroxisomes has been extensively examined. However, the supply of phospholipids, which is needed to generate peroxisomal membranes in mammals, remains unclear. Therefore, we herein investigated metabolic alterations induced by clofibric acid, a peroxisome proliferator, in the synthesis of phospholipids, particularly phosphatidylethanolamine (PE) molecular species, and their relationship with the biogenesis of peroxisomal membranes. The subcutaneous administration of clofibric acid to rats at a relatively low dose (130 mg/kg) once a day time-dependently and gradually increased the integrated perimeter of peroxisomes per 100 µm2 hepatocyte cytoplasm (PA). A strong correlation was observed between the content (µmol/mg DNA) of PE containing arachidonic acid (20:4) and PA (r2 = 0.9168). Moreover, the content of PE containing octadecenoic acid (18:1) positively correlated with PA (r2 = 0.8094). The treatment with clofibric acid markedly accelerated the formation of 16:0-20:4 PE by increasing the production of 20:4 and the activity of acyl chain remodeling of pre-existing PE molecular species. Increases in the acyl chain remodeling of PE by clofibric acid were mainly linked to the up-regulated expression of the Lpcat3 gene. On the other hand, clofibric acid markedly increased the formation of palmitic acid (16:0)-18:1 PE through de novo synthesis. These results suggest that the enhanced formation of particular PE molecular species is related to increases in the mass of peroxisomal membranes in peroxisome proliferation in the liver.


Assuntos
Ácido Araquidônico/biossíntese , Ácido Araquidônico/química , Ácido Clofíbrico/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Fígado/citologia , Peroxissomos/efeitos dos fármacos , Fosfatidiletanolaminas/química , Animais , Membranas Intracelulares/metabolismo , Masculino , Peroxissomos/metabolismo , Ratos , Ratos Wistar
12.
Mol Cell ; 81(13): 2693-2704.e12, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33964204

RESUMO

The assembly of nascent proteins into multi-subunit complexes is a tightly regulated process that must occur at high fidelity to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble cytosolic subunits to function. Here, we show that the kinase with no lysine 1 (WNK1), known for its role in hypertension and neuropathy, functions as an assembly factor for the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and directly competes for binding of E3 ubiquitin ligases, permitting assembly. Depletion of WNK1 thus destabilizes both the EMC and its membrane protein clients. This work describes an unexpected role for WNK1 in protein biogenesis and defines the general requirements of an assembly factor that will apply across the proteome.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Complexos Multiproteicos/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Retículo Endoplasmático/genética , Células HeLa , Humanos , Complexos Multiproteicos/genética , Proteína Quinase 1 Deficiente de Lisina WNK/genética
13.
FEBS Lett ; 595(14): 1902-1913, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34050946

RESUMO

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward-facing forms. The overall structure has a bilobate V-shape with pseudo-symmetrical assembly of the N- and C-lobes. In one crystal structure, the C-lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2-specific conformational transitions imply that an unprecedented movement of the transmembrane α-helix 7 is related to the release of the substrate into the vacuolar lumen.


Assuntos
Nicotina/química , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Plantas/química , Tabaco/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Modelos Moleculares , Nicotina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Células Vegetais/química , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tabaco/genética , Vacúolos/química
14.
Biochem Biophys Res Commun ; 561: 158-164, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34023781

RESUMO

Remodeling of vacuolar membranes mediated by endosomal sorting complex required for transport (ESCRT) is critical for microautophagy induction in budding yeast. Nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicit recruitment of the ESCRT-0 complex (Vps27-Hse1) onto vacuolar membranes and ESCRT-mediated microautophagy induction. Mitotic protein phosphatase Cdc14 antagonizes TORC1-mediated phosphorylation in macroautophagy induction after nutrient starvation and TORC1 inactivation. Here, we report that Cdc14 downregulates microautophagy induction after TORC1 inactivation. Cdc14 dysfunction stimulated the vacuolar membrane recruitment of Hse1, but not Vps27, after TORC1 inactivation, promoting ESCRT-0 complex formation. Conversely, overexpression of CDC14 compromises Hse1 recruitment on vacuolar membranes and microautophagy induction after TORC1 inactivation. Thus, Cdc14 phosphatase regulates the fluxes of two types of autophagy in the opposite directions, namely, it elicits macroautophagy and attenuates microautophagy.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Membranas Intracelulares/patologia , Microautofagia , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Vacúolos/patologia
15.
Nat Commun ; 12(1): 2616, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972548

RESUMO

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno , Retina/metabolismo , Fator de Resposta Sérica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Science ; 372(6545): 935-941, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33927055

RESUMO

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.


Assuntos
Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Legionella pneumophila/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas de Bactérias/química , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Retroalimentação Fisiológica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Membranas Intracelulares/ultraestrutura , Legionella pneumophila/enzimologia , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Mutação , Fosfatidilinositol 3-Quinase/química , Fosfatos de Fosfatidilinositol/química , Monoéster Fosfórico Hidrolases/metabolismo , Domínios Proteicos , Células RAW 264.7
17.
Dev Cell ; 56(10): 1452-1468.e8, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33878344

RESUMO

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-ß-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.


Assuntos
Autofagia , Axônios/metabolismo , Lipídeos/química , Lisossomos/metabolismo , Distrofias Musculares/patologia , Doença de Niemann-Pick Tipo C/patologia , Estresse Fisiológico , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Transporte Biológico , Morte Celular , Colesterol/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Membranas Intracelulares/metabolismo , Cinesina/metabolismo , Camundongos Endogâmicos BALB C , Distrofias Musculares/complicações , Proteína C1 de Niemann-Pick/deficiência , Proteína C1 de Niemann-Pick/metabolismo , Doença de Niemann-Pick Tipo C/complicações
18.
Cells ; 10(3)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806748

RESUMO

Plants are nonmotile life forms that are constantly exposed to changing environmental conditions during the course of their life cycle. Fluctuations in environmental conditions can be drastic during both day-night and seasonal cycles, as well as in the long term as the climate changes. Plants are naturally adapted to face these environmental challenges, and it has become increasingly apparent that membranes and their lipid composition are an important component of this adaptive response. Plants can remodel their membranes to change the abundance of different lipid classes, and they can release fatty acids that give rise to signaling compounds in response to environmental cues. Chloroplasts harbor the photosynthetic apparatus of plants embedded into one of the most extensive membrane systems found in nature. In part one of this review, we focus on changes in chloroplast membrane lipid class composition in response to environmental changes, and in part two, we will detail chloroplast lipid-derived signals.


Assuntos
Cloroplastos/metabolismo , Meio Ambiente , Membranas Intracelulares/metabolismo , Metabolismo dos Lipídeos , Lipídeos de Membrana/metabolismo , Plantas/metabolismo , Plantas/microbiologia
19.
Int J Mol Sci ; 22(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33799982

RESUMO

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson's disease and Kufor-Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Exocitose , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Mutação , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , ATPases Translocadoras de Prótons/genética , Espermina/metabolismo , Ubiquitina/metabolismo
20.
Plant Cell ; 33(2): 248-269, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793824

RESUMO

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.


Assuntos
Membranas Intracelulares/metabolismo , Fotossíntese , Análise de Sequência de RNA , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multiproteicos/metabolismo , Fotossíntese/genética , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Proteoma/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Tilacoides/metabolismo , Sistemas Toxina-Antitoxina , Transcriptoma/genética
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