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1.
Hum Mol Genet ; 33(14): 1195-1206, 2024 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-38621658

RESUMEN

Mutations in DNAJB6 are a well-established cause of limb girdle muscular dystrophy type D1 (LGMD D1). Patients with LGMD D1 develop progressive muscle weakness with histology showing fibre damage, autophagic vacuoles, and aggregates. Whilst there are many reports of LGMD D1 patients, the role of DNAJB6 in the muscle is still unclear. In this study, we developed a loss of function zebrafish model in order to investigate the role of Dnajb6. Using a double dnajb6a and dnajb6b mutant model, we show that loss of Dnajb6 leads to a late onset muscle weakness. Interestingly, we find that adult fish lacking Dnajb6 do not have autophagy or myofibril defects, however, they do show mitochondrial changes and damage. This study demonstrates that loss of Dnajb6 causes mitochondrial defects and suggests that this contributes to muscle weakness in LGMD D1. These findings expand our knowledge of the role of Dnajb6 in the muscle and provides a model to screen novel therapies for LGMD D1.


Asunto(s)
Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP40 , Mitocondrias , Chaperonas Moleculares , Debilidad Muscular , Distrofia Muscular de Cinturas , Pez Cebra , Animales , Humanos , Autofagia/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Mitocondrias/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/patología , Debilidad Muscular/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación , Proteínas del Tejido Nervioso , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
2.
EMBO J ; 41(19): e110398, 2022 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-35968799

RESUMEN

Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Agregado de Proteínas , Autofagia/fisiología , Endosomas/metabolismo , Lisosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo
3.
Mol Microbiol ; 121(4): 659-670, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38140856

RESUMEN

Since its inception in the 1930s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of <100 nm thick and with protozoans being larger than 1 µm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate, and speed up the structural characterisation of biological samples, offering new imaging modalities for the study of protozoans. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 µm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of protozoans.


Asunto(s)
Tomografía con Microscopio Electrónico , Microscopía Electrónica de Transmisión de Rastreo/métodos , Tomografía con Microscopio Electrónico/métodos , Microscopía por Crioelectrón/métodos , Microscopía Electrónica de Rastreo
4.
Kidney Int ; 105(1): 132-149, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38069998

RESUMEN

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.


Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ratas , Ratones , Animales , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/genética , Liraglutida/farmacología , Liraglutida/uso terapéutico , Receptor del Péptido 1 Similar al Glucagón/genética , Diabetes Mellitus Experimental/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/uso terapéutico , Inflamación
5.
Proteomics ; : e2300087, 2023 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-38059892

RESUMEN

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1α and IL-1ß. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

6.
Microcirculation ; 30(7): e12823, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37494581

RESUMEN

OBJECTIVE: The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. METHODS: We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. RESULTS: Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. CONCLUSIONS: These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.


Asunto(s)
Monocitos , Neutrófilos , Hialuronoglucosaminidasa , Células Endoteliales , Endotelio
7.
Int J Mol Sci ; 23(10)2022 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-35628580

RESUMEN

Over 50% of the world's population is infected with Human Cytomegalovirus (HCMV). HCMV is responsible for serious complications in the immuno-compromised and is a leading cause of congenital birth defects. The molecular function of many HCMV proteins remains unknown, and a deeper understanding of the viral effectors that modulate virion maturation is required. In this study, we observed that UL34 is a viral protein expressed with leaky late kinetics that localises to the nucleus during infection. Deletion of UL34 from the HCMV genome (ΔUL34) did not abolish the spread of HCMV. Instead, over >100-fold fewer infectious virions were produced, so we report that UL34 is an augmenting gene. We found that ΔUL34 is dispensable for viral DNA replication, and its absence did not alter the expression of IE1, MCP, gB, UL26, UL83, or UL99 proteins. In addition, ΔUL34 infections were able to progress through the replication cycle to form a viral assembly compartment; however, virion maturation in the cytoplasm was abrogated. Further examination of the nucleus in ΔUL34 infections revealed replication compartments with aberrant morphology, containing significantly less assembled capsids, with almost none undergoing subsequent maturation. Therefore, this work lays the foundation for UL34 to be further investigated in the context of nuclear organization and capsid maturation during HCMV infection.


Asunto(s)
Cápside , Citomegalovirus , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Citomegalovirus/genética , Citomegalovirus/metabolismo , Replicación del ADN , ADN Viral/metabolismo , Humanos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genética
8.
Int J Mol Sci ; 22(11)2021 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-34073041

RESUMEN

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.


Asunto(s)
Modelos Animales de Enfermedad , Hidrolasas/genética , Mucopolisacaridosis III , Animales , Humanos , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/patología , Mutación , Fenotipo , Pez Cebra
9.
J Neurosci ; 39(28): 5562-5580, 2019 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-31061088

RESUMEN

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxß in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxß displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1 Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.


Asunto(s)
Transporte Axonal , Encefalomielitis Autoinmune Experimental/metabolismo , Vaina de Mielina/metabolismo , Receptor Nogo 1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Axones/metabolismo , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinesinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Receptor Nogo 1/genética , Células Ganglionares de la Retina/metabolismo , Transducción de Señal
10.
PLoS Pathog ; 14(3): e1006945, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29601598

RESUMEN

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.


Asunto(s)
Apoptosis , Membrana Celular/metabolismo , Gonorrea/patología , Macrófagos/patología , Mitocondrias/patología , Neisseria gonorrhoeae/patogenicidad , Porinas/metabolismo , Animales , Gonorrea/microbiología , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/microbiología , Porinas/genética
11.
PLoS Biol ; 15(1): e2000731, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28125611

RESUMEN

Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC.


Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , Pulmón/citología , Células Madre/citología , Animales , Biomarcadores/metabolismo , Muerte Celular , Separación Celular , Roturas del ADN de Doble Cadena , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Neoplasias de Células Escamosas/patología , Alveolos Pulmonares/citología , Fumar/efectos adversos , Tráquea/citología
12.
Am J Hum Genet ; 99(5): 1086-1105, 2016 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-27745833

RESUMEN

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.


Asunto(s)
Núcleo Celular/genética , Miopatías Distales/genética , Variación Genética , Miopatías Estructurales Congénitas/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Estudios de Cohortes , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Citoplasma/metabolismo , Miopatías Distales/patología , Proteína 4 Similar a ELAV/genética , Proteína 4 Similar a ELAV/metabolismo , Femenino , Flavoproteínas/metabolismo , Eliminación de Gen , Estudio de Asociación del Genoma Completo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Células HEK293 , Humanos , Masculino , Músculo Esquelético/patología , Mutación Missense , Miopatías Estructurales Congénitas/patología , Oxidorreductasas/metabolismo , Linaje , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Pez Cebra/genética
13.
Hum Mol Genet ; 25(11): 2131-2142, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-26969713

RESUMEN

Myofibrillar myopathy is a progressive muscle disease characterized by the disintegration of muscle fibers and formation of protein aggregates. Causative mutations have been identified in nine genes encoding Z-disk proteins, including the actin binding protein filamin C (FLNC). To investigate the mechanism of disease in FLNCW2710X myopathy we overexpressed fluorescently tagged FLNC or FLNCW2710X in zebrafish. Expression of FLNCW2710X causes formation of protein aggregates but surprisingly, our studies reveal that the mutant protein localizes correctly to the Z-disk and is capable of rescuing the fiber disintegration phenotype that results from FLNC knockdown. This demonstrates that the functions necessary for muscle integrity are not impaired, and suggests that it is the formation of protein aggregates and subsequent sequestration of FLNC away from the Z-disk that results in myofibrillar disintegration. Similar to those found in patients, the aggregates in FLNCW2710X expressing fish contain the co-chaperone BAG3. FLNC is a target of the BAG3-mediated chaperone assisted selective autophagy (CASA) pathway and therefore we investigated its role, and the role of autophagy in general, in clearing protein aggregates. We reveal that despite BAG3 recruitment to the aggregates they are not degraded via CASA. Additionally, recruitment of BAG3 is sufficient to block alternative autophagy pathways which would otherwise clear the aggregates. This blockage can be relieved by reducing BAG3 levels or by stimulating autophagy. This study therefore identifies both BAG3 reduction and autophagy promotion as potential therapies for FLNCW2710X myofibrillar myopathy, and identifies protein insufficiency due to sequestration, compounded by impaired autophagy, as the cause.


Asunto(s)
Autofagia/genética , Filaminas/genética , Fibras Musculares Esqueléticas/patología , Miopatías Estructurales Congénitas/genética , Animales , Regulación de la Expresión Génica , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patología , Mutación , Miofibrillas/genética , Miofibrillas/patología , Fenotipo , Agregación Patológica de Proteínas/genética , Sarcómeros/genética , Sarcómeros/patología , Pez Cebra/genética
14.
Nephrology (Carlton) ; 23(9): 815-820, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-29504645

RESUMEN

The role of autophagy in the kidney and many nephrological diseases has gained prominence in recent years. Much of this research has been focused on markers of autophagy that are static and reveal little about the state of this dynamic pathway. Other mechanistic investigations are limited to in vitro studies, that often provide circumstantial evidence of autophagic flux. Here we describe a method for measuring autophagic flux ex vivo that allows more direct observations to be made in situ regarding the state of autophagic flux within the renal cortex of a single animal.


Asunto(s)
Autofagosomas/ultraestructura , Autofagia , Corteza Renal/ultraestructura , Microscopía Electrónica de Transmisión , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Regulación de la Expresión Génica , Técnicas In Vitro , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Macrólidos/farmacología , Masculino , Ratones Endogámicos C57BL , Transducción de Señal , Sirolimus/farmacología , Factores de Tiempo
15.
J Immunol ; 195(12): 5770-9, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26566675

RESUMEN

Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.


Asunto(s)
Actinas/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Citoesqueleto/inmunología , Neutrófilos/fisiología , Tetraspaninas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígenos CD18/metabolismo , Adhesión Celular , Movimiento Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiotaxis/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Tetraspaninas/genética , Proteína de Unión al GTP rac1/genética
16.
Diabetologia ; 59(2): 379-89, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26508318

RESUMEN

AIMS/HYPOTHESIS: Changes in podocyte morphology and function are associated with albuminuria and progression of diabetic nephropathy. NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and Nox4 is upregulated in podocytes in response to high glucose. We assessed the role of NOX4-derived ROS in podocytes in vivo in a model of diabetic nephropathy using a podocyte-specific NOX4-deficient mouse, with a major focus on the development of albuminuria and ultra-glomerular structural damage. METHODS: Streptozotocin-induced diabetes-associated changes in renal structure and function were studied in male floxedNox4 and podocyte-specific, NOX4 knockout (podNox4KO) mice. We assessed albuminuria, glomerular extracellular matrix accumulation and glomerulosclerosis, and markers of ROS and inflammation, as well as glomerular basement membrane thickness, effacement of podocytes and expression of the podocyte-specific protein nephrin. RESULTS: Podocyte-specific Nox4 deletion in streptozotocin-induced diabetic mice attenuated albuminuria in association with reduced vascular endothelial growth factor (VEGF) expression and prevention of the diabetes-induced reduction in nephrin expression. In addition, podocyte-specific Nox4 deletion reduced glomerular accumulation of collagen IV and fibronectin, glomerulosclerosis and mesangial expansion, as well as glomerular basement membrane thickness. Furthermore, diabetes-induced increases in renal ROS, glomerular monocyte chemoattractant protein-1 (MCP-1) and protein kinase C alpha (PKC-α) were attenuated in podocyte-specific NOX4-deficient mice. CONCLUSIONS/INTERPRETATION: Collectively, this study shows the deleterious effect of Nox4 expression in podocytes by promoting podocytopathy in association with albuminuria and extracellular matrix accumulation in experimental diabetes, emphasising the role of NOX4 as a target for new renoprotective agents.


Asunto(s)
Citoprotección/genética , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/genética , NADPH Oxidasas/genética , Podocitos/metabolismo , Albuminuria/genética , Albuminuria/patología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/patología , Eliminación de Gen , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Especificidad de Órganos/genética , Podocitos/patología , Estreptozocina
17.
Mol Microbiol ; 97(4): 616-29, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25976323

RESUMEN

In Gram-negative bacteria, ß-barrel proteins are integrated into the outer membrane by the ß-barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo-electron microscopy show a diverse set of secretion pores in Gram-negative bacteria, with α-helix (Wza and GspD) or ß-strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG ß-barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Sistemas de Secreción Bacterianos/metabolismo , Microscopía por Crioelectrón , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/genética , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Porinas/química , Porinas/genética , Porinas/metabolismo , Estructura Secundaria de Proteína
18.
Diabetologia ; 58(9): 2074-8, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26071760

RESUMEN

AIMS/HYPOTHESIS: Defective beta cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy. Whether this dysregulation represents augmentation or inhibition is unclear because of technical limitations in assaying autophagy. The current aim was to determine the effects of high-fat feeding on true autophagic flux in beta cells in vivo in mice, and to establish the relationship between autophagy, endoplasmic reticulum (ER) stress and apoptosis. METHODS: Green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) mice were fed chow or high-fat diets for 8-10 weeks and injected with 100 mg kg(-1) day(-1) chloroquine for 5 days, prior to being killed, to block clearance of autophagic markers. Pancreases and livers were fixed and GFP-LC3 aggregates or autophagosomes were detected by fluorescence or electron microscopy, respectively. Independently, islets isolated from chow or high-fat-fed mice were treated for 2 h with chloroquine ex vivo, and immunoblotting was performed for markers of autophagy (LC3 lipidation - LC3II and p62/SQSTM1), ER stress (C/EBP homology protein [CHOP], phosphorylated eukaryotic initiation factor 2α [p-eIFα] and inositol requiring enzyme 1α [p-IRE1α]) and apoptosis (cleaved caspase-3). RESULTS: Numbers of autophagosomes and GFP puncta were increased in beta cells by combined high-fat feeding and chloroquine injection, indicative of enhanced autophagic flux. By contrast, GFP puncta were attenuated in liver under the same conditions. Relative to chow-fed controls, islets isolated from fat-fed mice exhibited higher LC3II levels when treated ex vivo with chloroquine. The combination of high-fat feeding and acute chloroquine treatment induced CHOP, p-eIF2α and caspase-3, but not either treatment alone. CONCLUSIONS/INTERPRETATION: We provide the first in vivo demonstrations that high-fat feeding increases autophagic flux in pancreatic beta cells, and that this serves to protect against induction of terminal ER stress. We also highlight an approach for monitoring dietary alterations in autophagic flux using ex vivo manipulation of isolated islets.


Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Dieta Alta en Grasa , Células Secretoras de Insulina/citología , Animales , Apoptosis , Autofagia , Caspasa 3/metabolismo , Cloroquina/química , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Homocigoto , Lípidos/química , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Factor de Transcripción CHOP/metabolismo
19.
Acta Neuropathol ; 130(3): 389-406, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25931053

RESUMEN

Nemaline myopathy is characterized by muscle weakness and the presence of rod-like (nemaline) bodies. The genetic etiology of nemaline myopathy is becoming increasingly understood with mutations in ten genes now known to cause the disease. Despite this, the mechanism by which skeletal muscle weakness occurs remains elusive, with previous studies showing no correlation between the frequency of nemaline bodies and disease severity. To investigate the formation of nemaline bodies and their role in pathogenesis, we generated overexpression and loss-of-function zebrafish models for skeletal muscle α-actin (ACTA1) and nebulin (NEB). We identify three distinct types of nemaline bodies and visualize their formation in vivo, demonstrating these nemaline bodies not only exhibit different subcellular origins, but also have distinct pathological consequences within the skeletal muscle. One subtype is highly dynamic and upon breakdown leads to the accumulation of cytoplasmic actin contributing to muscle weakness. Examination of a Neb-deficient model suggests this mechanism may be common in nemaline myopathy. Another subtype results from a reduction of actin and forms a more stable cytoplasmic body. In contrast, the final type originates at the Z-disk and is associated with myofibrillar disorganization. Analysis of zebrafish and muscle biopsies from ACTA1 nemaline myopathy patients demonstrates that nemaline bodies also possess a different protein signature. In addition, we show that the ACTA1(D286G) mutation causes impaired actin incorporation and localization in the sarcomere. Together these data provide a novel examination of nemaline body origins and dynamics in vivo and identifies pathological changes that correlate with muscle weakness.


Asunto(s)
Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miopatías Nemalínicas/patología , Miopatías Nemalínicas/fisiopatología , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Citoplasma/metabolismo , Citoplasma/patología , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Morfolinos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Debilidad Muscular/patología , Debilidad Muscular/fisiopatología , Mutación , Fenotipo , Sarcómeros/metabolismo , Sarcómeros/patología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
20.
Helicobacter ; 20(4): 269-83, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25669590

RESUMEN

BACKGROUND: Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. METHODS: H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. RESULTS: H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. CONCLUSIONS: This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Interleucina-8/metabolismo , Proteínas Periplasmáticas/metabolismo , Membrana Celular/fisiología , Ensayo de Immunospot Ligado a Enzimas , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Espectrometría de Masas , Microscopía Electrónica de Rastreo , Proteínas Periplasmáticas/genética
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