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1.
Cell ; 187(8): 1907-1921.e16, 2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38552624

RESUMEN

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.


Asunto(s)
Chlamydomonas reinhardtii , Cilios , Glicoproteínas , Cilios/química , Glicoproteínas/química , Glicosilación , Hidroxiprolina/química , Plantas/metabolismo , Chlamydomonas reinhardtii/química
2.
Cell ; 183(1): 169-184.e13, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32931734

RESUMEN

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.


Asunto(s)
Infecciones por Coronavirus/inmunología , Inmunogenicidad Vacunal , Neumonía Viral/inmunología , Vacunas Virales/inmunología , Adenoviridae/genética , Administración Intranasal , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19 , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Humanos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Pandemias , Neumonía Viral/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero , Vacunas Virales/administración & dosificación
3.
Cell ; 179(4): 909-922.e12, 2019 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-31668805

RESUMEN

The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.


Asunto(s)
Axonema/ultraestructura , Cilios/ultraestructura , Ciliopatías/patología , Microtúbulos/ultraestructura , Axonema/química , Axonema/genética , Movimiento Celular/genética , Cilios/química , Cilios/genética , Ciliopatías/genética , Ciliopatías/metabolismo , Microscopía por Crioelectrón , Humanos , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/ultraestructura , Microtúbulos/química , Microtúbulos/genética , Estrés Mecánico
4.
EMBO Rep ; 25(4): 2045-2070, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38454159

RESUMEN

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.


Asunto(s)
Proteínas del Citoesqueleto , Infertilidad Masculina , Teratozoospermia , Tiazoles , Animales , Humanos , Masculino , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dineínas/metabolismo , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Semen/metabolismo , Cabeza del Espermatozoide/fisiología , Espermatogénesis/genética , Espermatozoides/metabolismo , Teratozoospermia/metabolismo , Teratozoospermia/patología
6.
Fish Shellfish Immunol ; 34(6): 1432-8, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23542606

RESUMEN

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene.


Asunto(s)
Carpas/genética , Proteínas de Peces/genética , Factor 1 Regulador del Interferón/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carpas/inmunología , Carpas/metabolismo , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Factor 1 Regulador del Interferón/química , Factor 1 Regulador del Interferón/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Poli I-C/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
bioRxiv ; 2023 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-38187686

RESUMEN

In large vertebrate spindles, the majority of microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation in the presence of TPX2, another branching-promoting factor, as HURP's localization to microtubules is enhanced by TPX2 condensation. Lastly, we provide a structure of HURP on the microtubule lattice, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

8.
Nat Commun ; 14(1): 2072, 2023 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-37055408

RESUMEN

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin's structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation.


Asunto(s)
Proteínas Asociadas a Microtúbulos , Microtúbulos , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Microscopía por Crioelectrón , Microtúbulos/metabolismo , Vertebrados/metabolismo , Sitios de Unión , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo
9.
Nat Commun ; 14(1): 7338, 2023 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-37957156

RESUMEN

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.


Asunto(s)
Autofagosomas , Autofagia , Autofagosomas/metabolismo , Autofagia/fisiología , Macroautofagia , Fagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Lípidos
10.
Fish Shellfish Immunol ; 33(1): 42-7, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22510210

RESUMEN

With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.


Asunto(s)
Carpas/genética , Carpas/metabolismo , Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Calor , Estrés Fisiológico , Animales , Carpas/clasificación , Línea Celular Tumoral , Células Cultivadas , Citrato (si)-Sintasa/genética , Frío , Escherichia coli/genética , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/química , Ratones , Datos de Secuencia Molecular , Mutación , Filogenia , Homología de Secuencia de Aminoácido , Estrés Fisiológico/genética
11.
Cell Res ; 32(7): 659-669, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35477997

RESUMEN

Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes.


Asunto(s)
Actinas , Autofagia , Actinas/metabolismo , Animales , Autofagia/fisiología , Ratones , Miosinas/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismo
12.
Nat Commun ; 12(1): 3065, 2021 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-34031406

RESUMEN

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of ß-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.


Asunto(s)
Microscopía por Crioelectrón , Interacciones Huésped-Parásitos/fisiología , Proteínas de Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Toxoplasma/metabolismo , Sitios de Unión , Sistemas CRISPR-Cas , Humanos , Proteínas de Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Paclitaxel/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
13.
Nat Struct Mol Biol ; 28(1): 29-37, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33318703

RESUMEN

In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin-dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.


Asunto(s)
Chlamydomonas reinhardtii/anatomía & histología , Cilios/metabolismo , Locomoción/fisiología , Proteínas de Plantas/metabolismo , Axonema/metabolismo , Fenómenos Biomecánicos/fisiología , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Nexinas de Clasificación/metabolismo
14.
Cell Rep ; 37(4): 109881, 2021 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-34655519

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Microscopía por Crioelectrón , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Humanos , Ratones , Dominios y Motivos de Interacción de Proteínas/inmunología , Estructura Cuaternaria de Proteína , Glicoproteína de la Espiga del Coronavirus/química
15.
Cell Res ; 30(6): 551-552, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32284560

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

16.
Cell Host Microbe ; 28(3): 465-474.e4, 2020 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-32798445

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections, and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin-converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice that expressed human ACE2 and were immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.


Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , Virus de la Estomatitis Vesicular Indiana/genética , Vacunas Virales/genética , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , COVID-19 , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Peptidil-Dipeptidasa A/genética , Neumonía Viral/inmunología , Neumonía Viral/virología , Receptores Virales/genética , SARS-CoV-2 , Investigación Biomédica Traslacional , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacología , Células Vero , Virus de la Estomatitis Vesicular Indiana/inmunología , Vacunas Virales/inmunología , Vacunas Virales/farmacología
17.
Cell Res ; 27(8): 989-1001, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28731030

RESUMEN

Phosphatidylinositol 3-phosphate (PI3P) plays essential roles in vesicular trafficking, organelle biogenesis and autophagy. Two class III phosphatidylinositol 3-kinase (PI3KC3) complexes have been identified in mammals, the ATG14L complex (PI3KC3-C1) and the UVRAG complex (PI3KC3-C2). PI3KC3-C1 is crucial for autophagosome biogenesis, and PI3KC3-C2 is involved in various membrane trafficking events. Here we report the cryo-EM structures of human PI3KC3-C1 and PI3KC3-C2 at sub-nanometer resolution. The two structures share a common L-shaped overall architecture with distinct features. EM examination revealed that PI3KC3-C1 "stands up" on lipid monolayers, with the ATG14L BATs domain and the VPS34 C-terminal domain (CTD) directly contacting the membrane. Biochemical dissection indicated that the ATG14L BATs domain is responsible for membrane anchoring, whereas the CTD of VPS34 determines the orientation. Furthermore, PI3KC3-C2 binds much more weakly than PI3KC3-C1 to both PI-containing liposomes and purified endoplasmic reticulum (ER) vesicles, a property that is specifically determined by the ATG14L BATs domain. The in vivo ER localization analysis indicated that the BATs domain was required for ER localization of PI3KC3. We propose that the different lipid binding capacity is the key factor that differentiates the functions of PI3KC3-C1 and PI3KC3-C2 in autophagy.


Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/química , Complejos Multienzimáticos/química , Complejos Multienzimáticos/ultraestructura , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , Estructura Cuaternaria de Proteína
19.
Autophagy ; 12(7): 1168-79, 2016 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-27171858

RESUMEN

Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons.


Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/fisiología , Beclina-1/metabolismo , Neuronas Dopaminérgicas/metabolismo , Mesencéfalo/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase III , Dopamina/metabolismo , Ratones Noqueados , Degeneración Nerviosa/patología , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/metabolismo
20.
Nat Cell Biol ; 17(9): 1112-23, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26237647

RESUMEN

A fundamental question regarding autophagosome formation is how the shape of the double-membrane autophagosomal vesicle is generated. Here we show that in mammalian cells assembly of an actin scaffold inside the isolation membrane (the autophagosomal precursor) is essential for autophagosomal membrane shaping. Actin filaments are depolymerized shortly after starvation and actin is assembled into a network within the isolation membrane. When formation of actin puncta is disrupted by an actin polymerization inhibitor or by knocking down the actin-capping protein CapZß, isolation membranes and omegasomes collapse into mixed-membrane bundles. Formation of actin puncta is PtdIns(3)P dependent, and inhibition of PtdIns(3)P formation by treating cells with the PI(3)K inhibitor 3-MA, or by knocking down Beclin-1, abolishes the formation of actin puncta. Binding of CapZ to PtdIns(3)P, which is enriched in omegasomes, stimulates actin polymerization. Our findings illuminate the mechanism underlying autophagosomal membrane shaping and provide key insights into how autophagosomes are formed.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Autofagia , Proteína CapZ/fisiología , Membranas Intracelulares/metabolismo , Fagosomas/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Células Cultivadas , Membranas Intracelulares/ultraestructura , Fagosomas/ultraestructura , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Ratas
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