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Background & Aims: The lymphatic system plays crucial roles in maintaining fluid balance and immune regulation. Studying the liver lymphatics has been considered challenging, as common lymphatic endothelial cell (LyEC) markers are expressed by other liver cells. Additionally, isolation of sufficient numbers of LyECs has been challenging because of their extremely low abundance (<0.01% of entire liver cell population) in a normal liver. Methods: Potential LyEC markers was identified using our published single-cell RNA sequencing (scRNA-seq) dataset (GSE147581) in mouse livers. Interleukin-7 (IL7) promoter-driven green fluorescent protein knock-in heterozygous mice were used for the validation of IL7 expression in LyECs in the liver, for the development of liver LyEC isolation protocol, and generating liver ischemia/reperfusion (I/R) injury. Scanning electron microscopy was used for the structural analysis of LyECs. Changes in LyEC phenotypes in livers of mice with I/R were determined by RNA-seq analysis. Results: Through scRNA-seq analysis, we have identified IL7 as an exclusive marker for liver LyECs, with no overlap with other liver cell types. Based on IL7 expression in liver LyECs, we have established an LyEC isolation method and observed distinct cell surface structures of LyECs with fenestrae and cellular pores (ranging from 100 to 400 nm in diameter). Furthermore, we identified LyEC genes that undergo alterations during I/R liver injuries. Conclusions: This study not only identified IL7 as an exclusively expressed gene in liver LyECs, but also enhanced our understanding of LyEC structures and demonstrated transcriptomic changes in injured livers. Impact and implications: Understanding the lymphatic system in the liver is challenging because of the absence of specific markers for liver LyEC. This study has identified IL7 as a reliable marker for LyECs, enabling the development of an effective method for their isolation, elucidating their unique cell surface structure, and identifying LyEC genes that undergo changes during liver damage. The development of IL7 antibodies for detecting it in human liver specimens will further advance our understanding of the liver lymphatic system in the future.
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Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes. We provided evidence that active proteasomes found within the lumen of phagosomes and endolysosomal vesicles locally generate antigenic peptides that can be directly loaded onto trafficking MHC-I molecules. However, the mechanism of active proteasome delivery to the endocytic compartments remained unknown. In this study, we demonstrate that phagosome-associated LC3A/B structures deliver proteasomes into subcellular compartments containing exogenous Ags and that autophagy drives TAP-independent, proteasome-dependent cross-presentation.
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Reactividad Cruzada , Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Presentación de Antígeno , Autofagosomas , Fagosomas/metabolismo , Antígenos de Histocompatibilidad Clase I , Antígenos , Proteínas de Transporte de Membrana/metabolismo , Péptidos/metabolismoRESUMEN
IMPORTANCE: Neurospora is a quintessential tip-growing organism, which is well known for packaging and longitudinal transport of tip-building blocks. Thus far, however, little attention has been paid to the co-essential process of reclamation, that is-taking apart of upstream, older structural elements, otherwise known as "autophagy". We are not yet prepared to set out the chemistry of that elaborate process, but its morphological start alone is worthy of attention. Carbon starvation triggers significant autophagic changes, beginning with prolific vacuolation along the plasma membrane, and eventual filling of 70% (or more) of cytoplasmic volume. Additionally, the Neurospora plasma membrane elaborates a variety of phagophores which themselves often look lytic. These have either dual enclosing membranes, like the familiar autophagosomes, can be doubled and have four wrapping membranes, or can be compounded with multiple membrane layers. These reclamation processes must be accommodated by the mechanism of tip growth.
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Neurospora crassa , Neurospora crassa/metabolismo , Autofagia , Membrana Celular/metabolismoRESUMEN
Although mitochondrial activity is critical for angiogenesis, its mechanism is not entirely clear. Here we show that mice with endothelial deficiency of any one of the three nuclear genes encoding for mitochondrial proteins, transcriptional factor (TFAM), respiratory complex IV component (COX10), or redox protein thioredoxin 2 (TRX2), exhibit retarded retinal vessel growth and arteriovenous malformations (AVM). Single-cell RNA-seq analyses indicate that retinal ECs from the three mutant mice have increased TGFß signaling and altered gene expressions associated with vascular maturation and extracellular matrix, correlating with vascular malformation and increased basement membrane thickening in microvesels of mutant retinas. Mechanistic studies suggest that mitochondrial dysfunction from Tfam, Cox10, or Trx2 depletion induces a mitochondrial localization and MAPKs-mediated phosphorylation of SMAD2, leading to enhanced ALK5-SMAD2 signaling. Importantly, pharmacological blockade of ALK5 signaling or genetic deficiency of SMAD2 prevented retinal vessel growth retardation and AVM in all three mutant mice. Our studies uncover a novel mechanism whereby mitochondrial dysfunction via the ALK5-SMAD2 signaling induces retinal vascular malformations, and have therapeutic values for the alleviation of angiogenesis-associated human retinal diseases.
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Malformaciones Arteriovenosas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Proteína Smad2 , Animales , Ratones , Malformaciones Arteriovenosas/genética , Malformaciones Arteriovenosas/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Fosforilación , Transducción de Señal , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Ixodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands-critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions. RESULTS: Using multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut. CONCLUSIONS: This study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick. Video Abstract.
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Borrelia burgdorferi , Ixodes , Enfermedad de Lyme , Microbiota , Animales , Borrelia burgdorferi/genética , Ixodes/microbiología , Enfermedad de Lyme/microbiología , Mamíferos , Ratones , Ninfa/microbiologíaRESUMEN
Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6-12 h, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on ultrastructure of the Golgi apparatus using electron microscopy and EM tomography. Our initial results on protein secretion showed that Retro-2 treatment does not significantly influence secretion of either small or large cargos. Ultra-structural study of the Golgi, however, revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3-5 h, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi structural proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure to a much greater extent than previously anticipated.
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COVID-19 , Aparato de Golgi , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , SARS-CoV-2 , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. ATP synthase c-ring was suggested to house the leak channel of mitochondrial permeability transition (mPT), which activates during excitotoxic ischemic insult. In this present study, we purified human c-ring from both eukaryotic and prokaryotic hosts to biophysically characterize its channel activity. We show that purified c-ring forms a large multi-conductance, voltage-gated ion channel that is inhibited by the addition of ATP synthase F1 subcomplex. In contrast, dissociation of F1 from FO occurs during excitotoxic neuronal death suggesting that the F1 constitutes the gate of the channel. mPT is known to dissipate the osmotic gradient across the inner membrane during cell death. We show that ATP synthase c-subunit knock down (KD) prevents the osmotic change in response to high calcium and eliminates large conductance, Ca2+ and CsA sensitive channel activity of mPT. These findings elucidate the gating mechanism of the ATP synthase c-subunit leak channel (ACLC) and suggest how ACLC opening is regulated by cell stress in a CypD-dependent manner.
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Proteínas de Transporte de Membrana Mitocondrial , ATPasas de Translocación de Protón Mitocondriales , Adenosina Trifosfato/metabolismo , Muerte Celular , Humanos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón/metabolismoRESUMEN
Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe-/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe-/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe-/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.
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Aneurisma de la Aorta Abdominal/patología , Macrófagos/patología , Fagocitos/patología , Angiotensina II/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta Abdominal/metabolismo , Apolipoproteínas E/metabolismo , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fagocitos/metabolismo , Tomografía Computarizada por Rayos X/métodosRESUMEN
The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.
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Aminoácidos/química , Dominios Proteicos , Antígeno gp100 del Melanoma/química , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Humanos , Melanosomas/metabolismo , Mutación , Pliegue de Proteína , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Cerebral cavernous malformations (CCMs) are vascular abnormalities that primarily occur in adulthood and cause cerebral hemorrhage, stroke, and seizures. CCMs are thought to be initiated by endothelial cell (EC) loss of any one of the three Ccm genes: CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10). Here we report that mice with a brain EC-specific deletion of Pdcd10 (Pdcd10BECKO) survive up to 6-12 months and develop bona fide CCM lesions in all regions of brain, allowing us to visualize the vascular dynamics of CCM lesions using transcranial two-photon microscopy. This approach reveals that CCMs initiate from protrusion at the level of capillary and post-capillary venules with gradual dissociation of pericytes. Microvascular beds in lesions are hyper-permeable, and these disorganized structures present endomucin-positive ECs and α-smooth muscle actin-positive pericytes. Caveolae in the endothelium of Pdcd10BECKO lesions are drastically increased, enhancing Tie2 signaling in Ccm3-deficient ECs. Moreover, genetic deletion of caveolin-1 or pharmacological blockade of Tie2 signaling effectively normalizes microvascular structure and barrier function with attenuated EC-pericyte disassociation and CCM lesion formation in Pdcd10BECKO mice. Our study establishes a chronic CCM model and uncovers a mechanism by which CCM3 mutation-induced caveolae-Tie2 signaling contributes to CCM pathogenesis.
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Proteínas Reguladoras de la Apoptosis/deficiencia , Encéfalo/metabolismo , Caveolas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Receptor TIE-2/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Encéfalo/patología , Encéfalo/ultraestructura , Caveolas/ultraestructura , Células Cultivadas , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Humanos , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Pericitos/metabolismo , Receptor TIE-2/genética , Transducción de Señal , Análisis de SupervivenciaRESUMEN
The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis.
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Acilcoenzima A/genética , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Homeostasis/genética , Humanos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase ß subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.
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Adenosina Trifosfato/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Subunidades de Proteína/metabolismo , Animales , Línea Celular , Ciclo del Ácido Cítrico/fisiología , Fibroblastos/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas/metabolismo , ARN Mensajero , Sinapsis/metabolismoRESUMEN
The Golgi is surrounded by a ribosome-excluding matrix. Recently, we reported that the cis-Golgi-localized golgin GM130 can phase-separate to form dynamic, liquid-like condensates in vitro and in vivo. Here, we show that the overexpression of each of the remaining cis (golgin160, GMAP210)- and trans (golgin97, golgin245, GCC88, GCC185)-golgins results in novel protein condensates. Focused ion beam scanning electron microscopy (FIB-SEM) images of GM130 condensates reveal a complex internal organization with branching aqueous channels. Pairs of golgins overexpressed in the same cell form distinct juxtaposed condensates. These findings support the hypothesis that, in addition to their established roles as vesicle tethers, phase separation may be a common feature of the golgin family that contributes to Golgi organization.
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Autoantígenos/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Autoantígenos/química , Autoantígenos/ultraestructura , Supervivencia Celular , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas de la Matriz de Golgi/química , Proteínas de la Matriz de Golgi/ultraestructura , Células HeLa , Humanos , Imagen de Lapso de Tiempo , Red trans-Golgi/metabolismoRESUMEN
Formation of a perfusable microvascular network (µVN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a µVN, but distinct stromal cell populations may play different roles in this process. Here we describe the differential effects that two widely used stromal cell populations, fibroblasts (FBs) and pericytes (PCs), have on µVN formation. We examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable µVNs in fibrin gels cast within a microfluidic chamber. ECs alone failed to fully assemble a perfusable µVN. Human lung FBs stimulated the formation of EC-lined µVNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF). Addition of recombinant HGF improved while the c-MET inhibitor, Capmatinib (INCB28060), reduced µVN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. Different stromal cell types provide different functions in microvessel assembly by ECs. FBs support µVN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology.
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The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.
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Proteínas Quinasas Activadas por AMP/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Microautofagia/genética , Complejo de la Endopetidasa Proteasomal/genética , Citoplasma/genética , Citoplasma/metabolismo , Glucosa/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/genética , Saccharomyces cerevisiae/genética , Inanición/genética , Inanición/metabolismo , Ubiquitina/genética , Ubiquitinación/genética , Vacuolas/genética , Vacuolas/metabolismoRESUMEN
The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn't lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.
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Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Proteínas Qa-SNARE/metabolismo , Sustitución de Aminoácidos , Aparato de Golgi/genética , Aparato de Golgi/ultraestructura , Proteínas de la Matriz de Golgi/genética , Células HeLa , Humanos , Microscopía Electrónica de Transmisión , Mutación Missense , Dominios Proteicos , Proteínas Qa-SNARE/genéticaRESUMEN
During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.
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Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/química , Complejo de la Endopetidasa Proteasomal/inmunología , Microglobulina beta-2/metabolismo , Animales , Presentación de Antígeno , Células Cultivadas , Reactividad Cruzada , Células Dendríticas/citología , Endocitosis , Humanos , Ratones , Fagosomas/inmunología , Proteolisis , Microglobulina beta-2/genéticaRESUMEN
Familial Parkinson's disease (PD) protein DJ-1 mutations are linked to early onset PD. We have found that DJ-1 binds directly to the F1FO ATP synthase ß subunit. DJ-1's interaction with the ß subunit decreased mitochondrial uncoupling and enhanced ATP production efficiency while in contrast mutations in DJ-1 or DJ-1 knockout increased mitochondrial uncoupling, and depolarized neuronal mitochondria. In mesencephalic DJ-1 KO cultures, there was a progressive loss of neuronal process extension. This was ameliorated by a pharmacological reagent, dexpramipexole, that binds to ATP synthase, closing a mitochondrial inner membrane leak and enhancing ATP synthase efficiency. ATP synthase c-subunit can form an uncoupling channel; we measured, therefore, ATP synthase F1 (ß subunit) and c-subunit protein levels. We found that ATP synthase ß subunit protein level in the DJ-1 KO neurons was approximately half that found in their wild-type counterparts, comprising a severe defect in ATP synthase stoichiometry and unmasking c-subunit. We suggest that DJ-1 enhances dopaminergic cell metabolism and growth by its regulation of ATP synthase protein components.
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Neuronas Dopaminérgicas/metabolismo , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Animales , Expresión Génica , Humanos , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , Unión Proteica , Proteína Desglicasa DJ-1/genética , Ratas Sprague-DawleyRESUMEN
The apicomplexan parasite Babesia microti is the primary agent of human babesiosis, a malaria-like illness and potentially fatal tick-borne disease. Unlike its close relatives, the agents of human malaria, B. microti develops within human and mouse red blood cells in the absence of a parasitophorous vacuole, and its secreted antigens lack trafficking motifs found in malarial secreted antigens. Here, we show that after invasion of erythrocytes, B. microti undergoes a major morphogenic change during which it produces an interlacement of vesicles (IOV); the IOV system extends from the plasma membrane of the parasite into the cytoplasm of the host erythrocyte. We developed antibodies against two immunodominant antigens of the parasite and used them in cell fractionation studies and fluorescence and immunoelectron microscopy analyses to monitor the mode of secretion of B. microti antigens. These analyses demonstrate that the IOV system serves as a major export mechanism for important antigens of B. microti and represents a novel mechanism for delivery of parasite effectors into the host by this apicomplexan parasite.
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Antígenos de Protozoos/inmunología , Babesia microti/inmunología , Babesia microti/metabolismo , Babesiosis/parasitología , Vesículas Transportadoras/metabolismo , Animales , Transporte Biológico , Modelos Animales de Enfermedad , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Humanos , Epítopos Inmunodominantes/inmunología , Ratones , Ratones NoqueadosRESUMEN
PMEL is a pigment cell-specific protein that forms a functional amyloid matrix in melanosomes. The matrix consists of well-separated fibrillar sheets on which the pigment melanin is deposited. Using electron tomography, we demonstrate that this sheet architecture is governed by the PMEL repeat (RPT) domain, which associates with the amyloid as an accessory proteolytic fragment. Thus, the RPT domain is dispensable for amyloid formation as such but shapes the morphology of the matrix, probably in order to maximize the surface area available for pigment adsorption. Although the primary amino acid sequence of the RPT domain differs vastly among various vertebrates, we show that it is a functionally conserved, interchangeable module. RPT domains of all species are predicted to be very highly O-glycosylated, which is likely the common defining feature of this domain. O-glycosylation is indeed essential for RPT domain function and the establishment of the PMEL sheet architecture. Thus, O-glycosylation, not amino acid sequence, appears to be the major factor governing the characteristic PMEL amyloid morphology.