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1.
Proc Natl Acad Sci U S A ; 117(31): 18530-18539, 2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32690699

RESUMEN

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.


Asunto(s)
Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Línea Celular , Retículo Endoplásmico/genética , Endosomas/genética , Endosomas/metabolismo , Humanos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
Proc Natl Acad Sci U S A ; 115(27): E6237-E6244, 2018 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-29915089

RESUMEN

The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets that is predominantly associated with the cell cortex in yeast. Upon treatment with rapamycin, the ER undergoes degradation by selective autophagy. This process, termed ER-phagy, requires Atg40, a selective autophagy receptor that localizes to the cortical ER. Here we report that ER-phagy also requires Lnp1, an ER membrane protein that normally resides at the three-way junctions of the ER network, where it serves to stabilize the network as it is continually remodeled. Rapamycin treatment increases the expression of Atg40, driving ER domains marked by Atg40 puncta to associate with Atg11, a scaffold protein needed to form autophagosomes. Although Atg40 largely localizes to the cortical ER, the autophagy machinery resides in the cell interior. The localization of Atg40 to sites of autophagosome formation is blocked in an lnp1Δ mutant or upon treatment of wild-type cells with the actin-depolymerizing drug Latrunculin A. This prevents the association of Atg40 with Atg11 and the packaging of the ER into autophagosomes. We propose that Lnp1 is needed to stabilize the actin-dependent remodeling of the ER that is essential for ER-phagy.


Asunto(s)
Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tiazolidinas/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
3.
J Biol Chem ; 292(29): 12111-12125, 2017 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-28576830

RESUMEN

Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , GTP Fosfohidrolasas/metabolismo , Leishmania donovani/metabolismo , Macrófagos/parasitología , Metaloendopeptidasas/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Línea Celular Tumoral , Citosol/enzimología , Citosol/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Leishmania donovani/citología , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Mutagénesis Sitio-Dirigida , Mutación , Organismos Modificados Genéticamente , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Factores de Virulencia/química , Factores de Virulencia/genética
4.
World J Microbiol Biotechnol ; 34(3): 45, 2018 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-29520519

RESUMEN

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.


Asunto(s)
Proteínas Bacterianas/farmacología , Citosol/metabolismo , Células Epiteliales/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/patogenicidad , Factores de Virulencia/metabolismo , Células A549 , Animales , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Femenino , Células HeLa/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Conejos , Elementos Reguladores de la Transcripción , Streptococcus agalactiae/genética , Virulencia/efectos de los fármacos , Factores de Virulencia/genética , Factores de Virulencia/aislamiento & purificación
5.
J Biol Chem ; 290(50): 29993-30005, 2015 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-26499792

RESUMEN

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.


Asunto(s)
Leishmania/enzimología , Proteínas de Unión al GTP rab1/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Datos de Secuencia Molecular , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab1/química
6.
Autophagy ; 20(9): 2067-2075, 2024 09.
Artículo en Inglés | MEDLINE | ID: mdl-38818751

RESUMEN

Reticulophagy is mediated by autophagy receptors that function in one of the two domains of the ER, tubules or flat sheets. Three different conserved mammalian receptors mediate autophagy in ER tubules: RTN3L, ATL3 and CALCOCO1. Previous studies have shown that RTN3L maintains proteostasis by targeting mutant aggregation-prone proteins for autophagy at distinct foci in ER tubules that we named ERPHS (ER-reticulophagy sites). The role for ATL3 and CALCOCO1 in proteostasis has not been addressed. Here we analyzed three different misfolded disease-causing RTN3L substrates and show that ATL3 and CALCOCO1 target the same cargoes for autophagy. Colocalization and knock down studies revealed that RTN3L and ATL3 are both required for the formation of RTN3L-containing ERPHS, while CALCOCO1 is not. We propose that RTN3L, ATL3 and CALCOCO1 work in parallel to maintain proteostasis within the ER network by targeting cargoes at different sites in the tubules.Abbreviation ATL3: atlastin GTPase 3; Baf: bafilomycin A1; CALCOCO1: calcium binding and coiled-coil domain 1; Epr1: ER-phagy receptor 1; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERPHS: ER-reticulophagy sites; LAMP1: lysosomal associated membrane protein 1; PGRMC1: progesterone receptor membrane component 1; POMC: proopiomelanocortin; Pro-AVP: pro-arginine vasopressin; RETREG1: reticulophagy regulator 1; reticulophagy: endoplasmic reticulum selective autophagy; RTN3L: reticulon 3 long isoform; VAPA: VAMP associated protein A.


Asunto(s)
Autofagia , Retículo Endoplásmico , Proteínas de la Membrana , Proteostasis , Humanos , Autofagia/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas , Proteínas de Unión al GTP/metabolismo , Células HeLa , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nogo
7.
Narra J ; 3(2): e147, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38454981

RESUMEN

Celiac disease (CD) is a gluten intolerance autoimmune disorder which its symptoms involve the gastrointestinal tract and sometimes the other organs. It is one of the most prevalent health problems rising in many populations as statistics show that in every 100 people about one person is suffering from CD. It has been observed that the persons who genetically contain the human leukocyte antigen (HLA) DQ2 and HLA DQ8 genes involved in the immune system haplotypes are more prone to develop an allergy to gluten. The only treatment currently available for CD is a strict gluten-free diet. However, recent research has shown promising new insights into the herbal-based treatments of CD. New insight on CD is now offering various prospects to manage its treatment, diagnosis, and serving in the development of advanced therapies. Several herbs and botanical extracts have demonstrated anti-inflammatory, immunomodulatory, and gut-healing properties that make them potential candidates for the management of CD. Here, we provide an updated review on pathogeneses and managements of CD. In particular, we summarize the current understandings of herbal-based treatments for CD and highlights their potential benefits.

8.
Autophagy ; 18(4): 937-938, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35100068

RESUMEN

The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets. When errors in protein folding occur, misfolded proteins accumulate in the ER. Proteostasis can be restored by ER quality control pathways. Reticulophagy is an ER quality control pathway that uses resident autophagy receptors to link an ER domain to the autophagy machinery. We recently showed that the reticulophagy receptor RTN3L recruits the COPII cargo adaptor SEC24C to target disease-causing mutant proinsulin INS2Akita puncta to the lysosome for degradation. When reticulophagy is disrupted and delivery to the lysosome is blocked, large INS2Akita puncta accumulate in the ER. Photobleach analysis revealed that these puncta behave like liquid condensates and not aggregates, as previously suggested. Other reticulophagy substrates that are segregated into tubules behave like INS2Akita, whereas a substrate of the ER sheets receptor, RETREG1/FAM134B, appears to be less fluid. Large INS2Akita puncta also accumulate when ER sheets are proliferated by the loss of LNPK, or by overproduction of the sheets-producing protein, CKAP4/CLIMP63. Restoring the tubular network by overexpressing reticulons reverses this phenotype. Our findings revealed that fluid-like deleterious cargoes are segregated into tubules to prevent them from expanding and affecting cell health while they are waiting to undergo reticulophagy.


Asunto(s)
Autofagia , Proteostasis , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Lisosomas/metabolismo
9.
Elife ; 102021 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-34467852

RESUMEN

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.


Asunto(s)
Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proinsulina/metabolismo , Animales , Autofagia , Línea Celular Tumoral , Células HEK293 , Humanos , Lisosomas , Ratones Noqueados , Agregado de Proteínas , Pliegue de Proteína
10.
Autophagy ; 16(2): 376-378, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31795809

RESUMEN

Endoplasmic reticulum (ER) homeostasis is maintained by the removal of misfolded ER proteins via different quality control pathways. Aggregation-prone proteins, including certain disease-linked proteins, are resistant to conventional ER degradation pathways and require other disposal mechanisms. Reticulophagy is a disposal pathway that uses resident autophagy receptors. How these receptors, which are dispersed throughout the ER network, target a specific ER domain for degradation is unknown. We recently showed in budding yeast, that ER stress upregulates the reticulophagy receptor, triggering its association with the COPII cargo adaptor complex, Sfb3/Lst1-Sec23 (SEC24C-SEC23 in mammals), to discrete sites on the ER. These domains are packaged into phagophores for degradation to prevent the accumulation of protein aggregates in the ER. This unconventional role for Sfb3/Lst1 is conserved in mammals and is independent of its role as a cargo adaptor on the secretory pathway. Our findings may have important therapeutic implications in protein-aggregation linked neurodegenerative disorders.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo
11.
Science ; 365(6448): 53-60, 2019 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-31273116

RESUMEN

The COPII-cargo adaptor complex Lst1-Sec23 selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that uses ER-resident receptors. Working in yeast, we found an unexpected role for Lst1-Sec23 in ER-phagy that was independent from its function in secretion. Up-regulation of the stress-inducible ER-phagy receptor Atg40 induced the association of Lst1-Sec23 with Atg40 at distinct ER domains to package ER into autophagosomes. Lst1-mediated ER-phagy played a vital role in maintaining cellular homeostasis by preventing the accumulation of an aggregation-prone protein in the ER. Lst1 function appears to be conserved because its mammalian homolog, SEC24C, was also required for ER-phagy.


Asunto(s)
Autofagia , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Estrés del Retículo Endoplásmico , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Respuesta de Proteína Desplegada
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