RESUMEN
Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.
Asunto(s)
Autofagia , Regulación hacia Abajo , Virus de la Encefalitis Japonesa (Especie) , Proteína 2 de la Membrana Asociada a los Lisosomas , Lisosomas , Lisosomas/metabolismo , Animales , Virus de la Encefalitis Japonesa (Especie)/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Porcinos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Encefalitis Japonesa/virología , Encefalitis Japonesa/veterinaria , Línea Celular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genéticaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease. Ginsenoside Rg2 has shown potential in treating AD, but the underlying protein regulatory mechanisms associated with ginsenoside Rg2 treatment for AD remain unclear. This study utilized scopolamine to induce memory impairment in mice, and proteomics methods were employed to investigate the potential molecular mechanism of ginsenoside Rg2 in treating AD model mice. The Morris water maze, hematoxylin and eosin staining, and Nissl staining results indicated that ginsenoside Rg2 enhanced cognitive ability and decreased neuronal damage in AD mice. Proteomics, western blot, and immunofluorescence results showed that ginsenoside Rg2 primarily improved AD mice by downregulating the expression of LGMN, LAMP1, and PSAP proteins through the regulation of the lysosomal pathway. Transmission electron microscopy and network pharmacology prediction results showed a potential connection between the mechanism of ginsenoside Rg2 treatment for AD mice and lysosomes. The comprehensive results indicated that ginsenoside Rg2 may improve AD by downregulating LGMN, LAMP1, and PSAP through the regulation of the lysosomal pathway.
Asunto(s)
Ginsenósidos , Lisosomas , Trastornos de la Memoria , Proteómica , Escopolamina , Animales , Ginsenósidos/farmacología , Ginsenósidos/administración & dosificación , Ratones , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Escopolamina/efectos adversos , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/inducido químicamente , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.
Asunto(s)
Autofagia , Complejo Dinactina , Lisosomas , Animales , Humanos , Ratones , Complejo 2 de Proteína Adaptadora/metabolismo , Autofagosomas/metabolismo , Complejo Dinactina/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neuronas/metabolismo , Unión Proteica , Sirolimus/farmacologíaRESUMEN
Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.
Asunto(s)
Apoptosis , Vesículas Extracelulares , Tetraspanina 30 , Proteína p53 Supresora de Tumor , Humanos , Tetraspanina 30/metabolismo , Tetraspanina 30/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Células HEK293 , Línea Celular Tumoral , Proliferación Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the preimplantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers connexin-43, insulin like growth factor binding protein 1 (IGFBP1), and prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on extracellular matrix (ECM) remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy.NEW & NOTEWORTHY Endocrine disruptors, such as para-nonylphenol, affect the decidualization of human endometrial stromal cells with an impact on decidualization itself, lysosome biogenesis and exocytosis, and extracellular matrix remodeling. All these alterations may negatively impact embryo implantation with the success of reproduction and the establishment of pregnancy.
Asunto(s)
Endometrio , Lisosomas , Fenoles , Prolactina , Células del Estroma , Humanos , Femenino , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/efectos de los fármacos , Fenoles/farmacología , Fenoles/toxicidad , Endometrio/metabolismo , Endometrio/efectos de los fármacos , Endometrio/citología , Prolactina/metabolismo , Decidua/metabolismo , Decidua/efectos de los fármacos , Decidua/citología , Exocitosis/efectos de los fármacos , Implantación del Embrión/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Embarazo , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.
Asunto(s)
Factor de Transcripción Activador 6 , Estrés del Retículo Endoplásmico , Lisosomas , Serina-Treonina Quinasas TOR , Tapsigargina , Proteína p53 Supresora de Tumor , Respuesta de Proteína Desplegada , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Serina-Treonina Quinasas TOR/metabolismo , Autofagia Mediada por Chaperones/efectos de los fármacos , Autofagia Mediada por Chaperones/genética , Mutación/genética , Línea Celular Tumoral , Autofagia/efectos de los fármacos , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Tunicamicina/farmacología , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.
Asunto(s)
Basigina , Neoplasias de la Mama , Matriz Extracelular , Proteína 1 de la Membrana Asociada a los Lisosomas , Metaloproteinasa 14 de la Matriz , Transportadores de Ácidos Monocarboxílicos , Invasividad Neoplásica , Podosomas , Femenino , Humanos , Basigina/metabolismo , Basigina/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Invasividad Neoplásica/genética , Podosomas/metabolismoRESUMEN
To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.
Asunto(s)
Burkholderia , Sistemas de Secreción Tipo VI , Burkholderia/metabolismo , Burkholderia/fisiología , Sistemas de Secreción Tipo VI/metabolismo , Humanos , Membrana Celular/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas Bacterianas/metabolismo , Actinas/metabolismo , Dinamina II/metabolismo , Autofagia , Galectinas/metabolismo , Interacciones Huésped-Patógeno , Extensiones de la Superficie Celular/metabolismo , Animales , Proteínas Asociadas a Microtúbulos , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
Acinetobacter baumannii (A. baumannii) causes autophagy flux disorder by degrading STX17, resulting in a serious inflammatory response. It remains unclear whether STX17 can alter the inflammatory response process by controlling autolysosome function. This study aimed to explore the role of STX17 in the regulation of pyroptosis induced by A. baumannii. Our findings indicate that overexpression of STX17 enhances autophagosome degradation, increases LAMP1 expression, reduces Cathepsin B release, and improves lysosomal function. Conversely, knockdown of STX17 suppresses autophagosome degradation, reduces LAMP1 expression, augments Cathepsin B release, and accelerates lysosomal dysfunction. In instances of A. baumannii infection, overexpression of STX17 was found to improve lysosomal function and reduce the expression of mature of GSDMD and IL-1ß, along with the release of LDH, thus inhibiting pyroptosis caused by A. baumannii. Conversely, knockdown of STX17 led to increased lysosomal dysfunction and further enhanced the expression of mature of GSDMD and IL-1ß, and increased the release of LDH, exacerbating pyroptosis induced by A. baumannii. These findings suggest that STX17 regulates pyroptosis induced by A. baumannii by modulating lysosomal function.
Asunto(s)
Acinetobacter baumannii , Interleucina-1beta , Lisosomas , Piroptosis , Proteínas Qa-SNARE , Lisosomas/metabolismo , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Humanos , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/genética , Autofagia , Animales , Catepsina B/metabolismo , Catepsina B/genética , Infecciones por Acinetobacter/microbiología , Ratones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Autofagosomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , GasderminasRESUMEN
Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles.
Asunto(s)
Neoplasias de la Mama , Proteína 1 de la Membrana Asociada a los Lisosomas , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Glicosilación , Femenino , Proteínas de Membrana de los Lisosomas/metabolismo , Espectrometría de Masas en Tándem , Polisacáridos/metabolismo , Polisacáridos/químicaRESUMEN
Entosis is a process that leads to the formation of cell-in-cell structures commonly found in cancers. Here, we identified entosis in hepatocellular carcinoma and the loss of Rnd3 (also known as RhoE) as an efficient inducer of this mechanism. We characterized the different stages and the molecular regulators of entosis induced after Rnd3 silencing. We demonstrated that this process depends on the RhoA/ROCK pathway, but not on E-cadherin. The proteomic profiling of entotic cells allowed us to identify LAMP1 as a protein upregulated by Rnd3 silencing and implicated not only in the degradation final stage of entosis, but also in the full mechanism. Moreover, we found a positive correlation between the presence of entotic cells and the metastatic potential of tumors in human patient samples. Altogether, these data suggest the involvement of entosis in liver tumor progression and highlight a new perspective for entosis analysis in medicine research as a novel therapeutic target.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Entosis , Proteómica , Factores de Transcripción , Proteínas de Unión al GTP rho , Proteína 1 de la Membrana Asociada a los LisosomasRESUMEN
Autophagy is a major catabolic degradation and recycling process that maintains homeostasis in cells and is especially important in postmitotic neurons. We implemented a high-content phenotypic assay to discover small molecules that promote autophagic flux and completed target identification and validation studies to identify protein targets that modulate the autophagy pathway and promote neuronal health and survival. Efficient syntheses of the prioritized compounds were developed to readily access analogues of the initial hits, enabling initial structure-activity relationship studies to improve potency and preparation of a biotin-tagged pulldown probe that retains activity. This probe facilitated target identification and validation studies through pulldown and competition experiments using both an unbiased proteomics approach and western blotting to reveal Lamin A/C and LAMP1 as the protein targets of compound RH1115. Evaluation of RH1115 in neurons revealed that this compound induces changes to LAMP1 vesicle properties and alters lysosome positioning. Dysfunction of the autophagy-lysosome pathway has been implicated in a variety of neurodegenerative diseases, including Alzheimer's disease, highlighting the value of new strategies for therapeutic modulation and the importance of small-molecule probes to facilitate the study of autophagy regulation in cultured neurons and in vivo.
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Enfermedad de Alzheimer , Lamina Tipo A , Humanos , Lamina Tipo A/metabolismo , Autofagia/fisiología , Neuronas/metabolismo , Lisosomas/metabolismo , Enfermedad de Alzheimer/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismoRESUMEN
Introduction: Most cases of Merkel cell carcinoma (MCC), a rare and highly aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of oncoproteins including a truncated form of the viral large T antigen (LT) in infected cells. These oncoproteins are an attractive target for a therapeutic cancer vaccine. Methods: We designed a cancer vaccine that promotes potent, antigen-specific CD4 T cell responses to MCPyV-LT. To activate antigen-specific CD4 T cells in vivo, we utilized our nucleic acid platform, UNITE™ (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and potent antigen-specific T cell responses. LTS220A, encoding a mutated form of MCPyV-LT that diminishes its pro-oncogenic properties, was introduced into the UNITE™ platform. Results: Vaccination with LTS220A-UNITE™ DNA vaccine (ITI-3000) induced antigen-specific CD4 T cell responses and a strong humoral response that were sufficient to delay tumor growth of a B16F10 melanoma line expressing LTS220A. This effect was dependent on the CD4 T cells' ability to produce IFNγ. Moreover, ITI-3000 induced a favorable tumor microenvironment (TME), including Th1-type cytokines and significantly enhanced numbers of CD4 and CD8 T cells as well as NK and NKT cells. Additionally, ITI-3000 synergized with an α-PD-1 immune checkpoint inhibitor to further slow tumor growth and enhance survival. Conclusions: These findings strongly suggest that in pre-clinical studies, DNA vaccination with ITI-3000, using the UNITE™ platform, enhances CD4 T cell responses to MCPyV-LT that result in significant anti-tumor immune responses. These data support the initiation of a first-in-human (FIH) Phase 1 open-label study to evaluate the safety, tolerability, and immunogenicity of ITI-3000 in patients with polyomavirus-positive MCC (NCT05422781).
Asunto(s)
Vacunas contra el Cáncer , Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Neoplasias Cutáneas , Humanos , Antígenos Virales de Tumores/genética , Linfocitos T CD4-Positivos , Proteína 1 de la Membrana Asociada a los Lisosomas , Neoplasias Cutáneas/terapia , Microambiente Tumoral , Proteínas de Membrana de los LisosomasRESUMEN
Brain lower grade glioma (LGG) is a common type of glioma. The current treatment methods still have some limitations, and some LGG patients will inevitably continue to deteriorate after treatment. We found the value of lysosomal associated membrane proteins (LAMPs) in the diagnosis and prognosis of LGG, which helps to enhance the clinical understanding of LGG treatment and improved prognosis. We assess the role of LAMPs in LGG, via the publicly available TCGA database. We explored expression levels of LAMPs in LGG using GEPIA2, cBioPortal, and UALCAN databases. The correction of LAMPs expression levels with immune cell infiltration in LGG patient was assessed by TIMER database. The Lysosomal associated membrane protein 1 (LAMP1)/2/4 mRNA levels were significantly higher in LGG patients than in healthy controls. Morover, high mRNA expressions of LAMP1/2/Lysosomal associated membrane protein 3 were associated with poor overall survival. We found that the immune invasion of LGG was almost significantly correlated with the expression of LAMPs. The results suggested that mRNA expressions of LAMP1 and LAMP4 were significantly associated with histological subtypes in LGG patients. lysosomal associated membrane protein 2 and LAMP5 were significantly down-regulated expression in samples of TP53 mutant in LGG compared to TP53 wild type. In addition, Lysosomal associated membrane protein 3 and LAMP4 were significantly overexpressed in samples of TP53 mutant in LGG Enrichment analysis applied to each component indicated that biological function was primarily associated with series of pathways in synapse and immunity.
Asunto(s)
Glioma , Proteína 3 de la Membrana Asociada a Lisosoma , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas , Pronóstico , Encéfalo , Glioma/diagnóstico , Glioma/genética , Proteínas de Membrana de los Lisosomas/genéticaRESUMEN
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that usually manifests in childhood and is thought to be caused by a complex interaction of genetic, environmental, and immune factors. The majority of current ASD diagnostic methods rely on subjective behavioral observation and scale assessment, making early detection difficult. In this study, we confirmed that lysosomal-associated membrane protein 1 (LAMP1), a functional marker of immune cell activation and cytotoxic degranulation, was upregulated in ASD blood, brain cortex, and various genetic animal models or cells using bioinformatics approaches. The prognostic value of LAMP1 was investigated by correlating its expression with clinical ASD rating scales, and the receiver operating characteristic (ROC) curve analysis in ASD also revealed that it has a favorable diagnostic ability in distinguishing ASD from control cohort. According to gene set enrichment analysis (GSEA) results, LAMP1 correlated with genes that were enriched in natural kill and T cell immune function. Taking all of the evidence into account, we discovered that abnormal elevations of LAMP1 mRNA and protein in the blood of ASD children, may influence the development of ASD through its involvement in immune cell activity regulation. This report highlights a novel marker for ASD early detection as well as potential therapeutic targets.
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Trastorno del Espectro Autista , Animales , Proteína 1 de la Membrana Asociada a los Lisosomas , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/genética , Pronóstico , Biomarcadores , Factores de Transcripción , Biología ComputacionalRESUMEN
Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. Ultimately, after particle degradation, phagosomes then fragment to reform lysosomes through phagosome resolution. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation and resolution. These changes can be assessed at the single-phagosome level by using immunofluorescence methods. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Commonly, progression of phagosomes into phagolysosomes can be determined by staining cells for Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome by microscopy or flow cytometry. However, this method can be used to detect any molecular marker for which there are compatible antibodies for immunofluorescence.
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Fagocitosis , Fagosomas , Fagosomas/metabolismo , Macrófagos/metabolismo , Lisosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismoRESUMEN
Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.
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Lisosomas , Hormonas Tiroideas , Animales , Ratones , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Hormonas Tiroideas/metabolismo , Autofagia/fisiologíaRESUMEN
Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.
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Neuraminidasa , Rabdomiosarcoma , Animales , Haploinsuficiencia , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas , Lisosomas/metabolismo , Ratones , Neuraminidasa/genética , Neuraminidasa/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Microambiente TumoralRESUMEN
Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.
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Fiebre de Lassa , Virus Lassa , Dilatación , Distroglicanos/metabolismo , Distroglicanos/farmacología , Endosomas/metabolismo , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Internalización del VirusRESUMEN
OBJECTIVES: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans. METHODS: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8-16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m2) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting. RESULTS: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast . CONCLUSION: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss.