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1.
Anal Chim Acta ; 1302: 342506, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38580410

RESUMO

BACKGROUND: Mitophagy plays indispensable roles in maintaining intracellular homeostasis in most eukaryotic cells by selectively eliminating superfluous components or damaged organelles. Thus, the co-operation of mitochondrial probes and lysosomal probes was presented to directly monitor mitophagy in dual colors. Nowadays, most of the lysosomal probes are composed of groups sensitive to pH, such as morpholine, amine and other weak bases. However, the pH in lysosomes would fluctuate in the process of mitophagy, leading to the optical interference. Thus, it is crucial to develop a pH-insensitive probe to overcome this tough problem to achieve exquisite visualization of mitophagy. RESULTS: In this study, we rationally prepared a pH-independent lysosome probe to reduce the optical interference in mitophagy, and thus the process of mitophagy could be directly monitored in dual color through cooperation between IVDI and MTR, depending on Förster resonance energy transfer mechanism. IVDI shows remarkable fluorescence enhancement toward the increase of viscosity, and the fluorescence barely changes when pH varies. Due to the sensitivity to viscosity, the probe can visualize micro-viscosity alterations in lysosomes without washing procedures, and it showed better imaging properties than LTR. Thanks to the inertia of IVDI to pH, IVDI can exquisitely monitor mitophagy with MTR by FRET mechanism despite the changes of lysosomal pH in mitophagy, and the reduced fluorescence intensity ratio of green and red channels can indicate the occurrence of mitophagy. Based on the properties mentioned above, the real-time increase of micro-viscosity in lysosomes during mitophagy was exquisitely monitored through employing IVDI. SIGNIFICANCE AND NOVELTY: Compared with the lysosomal fluorescent probes sensitive to pH, the pH-inert probe could reduce the influence of pH variation during mitophagy to achieve exquisite visualization of mitophagy in real-time. Besides, the probe could monitor the increase of lysosomal micro-viscosity in mitophagy. So, the probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.


Assuntos
Corantes Fluorescentes , Mitofagia , Humanos , Concentração de Íons de Hidrogênio , Viscosidade , Células HeLa , Corantes Fluorescentes/química , Lisossomos/química
2.
Cell Mol Life Sci ; 81(1): 171, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38597989

RESUMO

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.


Assuntos
Conexina 43 , Ubiquitina-Proteína Ligases , Humanos , Conexina 43/genética , Ubiquitina-Proteína Ligases/genética , Junções Comunicantes , Lisossomos , Conexinas , Comunicação Celular
3.
Sci Adv ; 10(14): eadl5012, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38569033

RESUMO

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Proteínas do Envelope Viral/metabolismo , Fator de Transcrição AP-1/metabolismo , Pandemias , Replicação Viral , Lisossomos/metabolismo , Fatores de Ribosilação do ADP/metabolismo
4.
Int J Biol Sci ; 20(5): 1905-1926, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481802

RESUMO

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.


Assuntos
Nefropatias , Obstrução Ureteral , ATPases Vacuolares Próton-Translocadoras , Animais , Humanos , Camundongos , Apoptose , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Fibrose , Pontos de Checagem da Fase G2 do Ciclo Celular , Nefropatias/metabolismo , Lisossomos/metabolismo , Proteínas SNARE/metabolismo , Proteínas SNARE/farmacologia , Obstrução Ureteral/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/farmacologia
5.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474091

RESUMO

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.


Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-38488191

RESUMO

Lysosomes play a central role in biochemical signal transduction and oxidative stress in cells. Inducing lysosome membrane penetration (LMP) to cause lysosomal-dependent cell death (LCD) in tumor cells is an effective strategy for cancer therapy. Chemical drugs can destroy the stability of lysosomes by neutralizing protons within the lysosomes or enhancing the fragility of the lysosomal membranes. However, there remain several unsolved problems of traditional drugs in LMP induction due to insufficient lysosomal targeting, fast metabolism, and toxicity in normal cells. With the development of nanotechnology, magnetic nanoparticles have been demonstrated to target lysosomes naturally, providing a versatile tool for lysosomal modulation. Combined with excellent tissue penetration and spatiotemporal manipulability of magnetic fields, magnetic modulation of lysosomes progresses rapidly in inducing LMP and LCD for cancer therapy. This review comprehensively discussed the strategies of magnetic modulation of lysosomes for cancer therapy. The intrinsic mechanisms of LMP-induced LCD were first introduced. Then, the modulation of lysosomes by diverse physical outputs of magnetic fields was emphatically discussed. Looking forward, this review will shed the light on the prospect of magnetic modulation of lysosomes, inspiring future research of magnetic modulation strategy in cancer therapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.


Assuntos
Membranas Intracelulares , Neoplasias , Humanos , Morte Celular/fisiologia , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fenômenos Magnéticos
7.
Comput Biol Med ; 171: 108201, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38428097

RESUMO

BACKGROUND: Lysosomes serve as regulatory hubs, and play a pivotal role in human diseases. However, the precise functions and mechanisms of action of lysosome-related genes remain unclear in preeclampsia and cancers. This study aimed to identify lysosome-related biomarkers in preeclampsia, and further explore the biomarkers shared between preeclampsia and cancers. MATERIALS AND METHODS: We obtained GSE60438 and GSE75010 datasets from the Gene Expression Omnibus database, pre-procesed them and merged them into a training cohort. The limma package in R was used to identify the differentially expressed mRNAs between the preeclampsia and normal control groups. Differentially expressed lysosome-related genes were identified by intersecting the differentially expressed mRNAs and lysosome-related genes obtained from Gene Ontology and GSEA databases. Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed using the DAVID database. The CIBERSORT method was used to analyze immune cell infiltration. Weighted gene co-expression analyses and three machine learning algorithm were used to identify lysosome-related diagnostic biomarkers. Lysosome-related diagnostic biomarkers were further validated in the testing cohort GSE25906. Nomogram diagnostic models for preeclampsia were constructed. In addition, pan-cancer analysis of lysosome-related diagnostic biomarkers were identified by was performed using the TIMER, Sangebox and TISIDB databases. Finally, the Drug-Gene Interaction, TheMarker and DSigDB Databases were used for drug-gene interactions analysis. RESULTS: A total of 11 differentially expressed lysosome-related genes were identified between the preeclampsia and control groups. Three molecular clusters connected to lysosome were identified, and enrichment analysis demonstrated their strong relevance to the development and progression of preeclampsia. Immune infiltration analysis revealed significant immunity heterogeneity among different clusters. GBA, OCRL, TLR7 and HEXB were identified as lysosome-related diagnostic biomarkers with high AUC values, and validated in the testing cohort GSE25906. Nomogram, calibration curve, and decision curve analysis confirmed the accuracy of predicting the occurrence of preeclampsia based on OCRL and HEXB. Pan-cancer analysis showed that GBA, OCRL, TLR7 and HEXB were associated with the prognosis of patients with various tumors and tumor immune cell infiltration. Twelve drugs were identified as potential drugs for the treatment of preeclampsia and cancers. CONCLUSION: This study identified GBA, OCRL, TLR7 and HEXB as potential lysosome-related diagnostic biomarkers shared between preeclampsia and cancers.


Assuntos
Neoplasias , Pré-Eclâmpsia , Feminino , Gravidez , Humanos , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/genética , Receptor 7 Toll-Like , Lisossomos/genética , Biomarcadores , Biologia Computacional , Aprendizado de Máquina , Neoplasias/diagnóstico , Neoplasias/genética
8.
Cell Commun Signal ; 22(1): 165, 2024 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-38448982

RESUMO

BACKGROUND: Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. METHODS: Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. RESULTS: We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. CONCLUSIONS: Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Feminino , Humanos , Lisossomos , Neoplasias Ovarianas/tratamento farmacológico , Mitocôndrias , Cisplatino/farmacologia
9.
Anal Chim Acta ; 1297: 342303, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38438223

RESUMO

Nitric oxide (NO) plays critical roles in both physiology and pathology, serving as a significant signaling molecule. Recent investigations have uncovered the pivotal role of lysosome as a critical organelle where intracellular NO exists and takes function. In this study, we developed a novel ratiometric fluorescent probe called XL-NO and modified it with a morpholine unit, which followed the intramolecular charge transfer (ICT) mechanism. The probe could detect lysosomal nitric oxide with high selectivity and sensitivity. The probe XL-NO contained a secondary amine moiety that could readily react with NO in lysosomes, leading to the formation of the N-nitrosation product. The N-nitroso structure enhanced the capability in push-pull electron, which obviously led to the change of fluorescence from 621 nm to 521 nm. In addition, XL-NO was discovered to have some evident advantages, such as significant ratiometric signal (I521/I621) change, strong anti-interference ability, good biocompatibility, and a low detection limit (LOD = 44.3 nM), which were crucial for the detection of lysosomal NO. To evaluate the practical application of XL-NO, NO imaging experiments were performed in both living cells and zebrafish. The results from these experiments confirmed the feasibility and reliability of XL-NO for exogenous/endogenous NO imaging and lysosome targeting.


Assuntos
Corantes Fluorescentes , Óxido Nítrico , Animais , Reprodutibilidade dos Testes , Peixe-Zebra , Lisossomos
10.
Cancer Lett ; 587: 216728, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38431036

RESUMO

Lysosomes are crucial organelles responsible for the degradation of cytosolic materials and bulky organelles, thereby facilitating nutrient recycling and cell survival. However, lysosome also acts as an executioner of cell death, including ferroptosis, a distinctive form of regulated cell death that hinges on iron-dependent phospholipid peroxidation. The initiation of ferroptosis necessitates three key components: substrates (membrane phospholipids enriched with polyunsaturated fatty acids), triggers (redox-active irons), and compromised defence mechanisms (GPX4-dependent and -independent antioxidant systems). Notably, iron assumes a pivotal role in ferroptotic cell death, particularly in the context of cancer, where iron and oncogenic signaling pathways reciprocally reinforce each other. Given the lysosomes' central role in iron metabolism, various strategies have been devised to harness lysosome-mediated iron metabolism to induce ferroptosis. These include the re-mobilization of iron from intracellular storage sites such as ferritin complex and mitochondria through ferritinophagy and mitophagy, respectively. Additionally, transcriptional regulation of lysosomal and autophagy genes by TFEB enhances lysosomal function. Moreover, the induction of lysosomal iron overload can lead to lysosomal membrane permeabilization and subsequent cell death. Extensive screening and individually studies have explored pharmacological interventions using clinically available drugs and phytochemical agents. Furthermore, a drug delivery system involving ferritin-coated nanoparticles has been specifically tailored to target cancer cells overexpressing TFRC. With the rapid advancements in understandings the mechanistic underpinnings of ferroptosis and iron metabolism, it is increasingly evident that lysosomes represent a promising target for inducing ferroptosis and combating cancer.


Assuntos
Ferro , Neoplasias , Humanos , Morte Celular , Ferro/metabolismo , Ferritinas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Lisossomos/metabolismo
11.
Biochem Pharmacol ; 222: 116111, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458329

RESUMO

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.


Assuntos
Autofagia , Neoplasias da Bexiga Urinária , Humanos , Autofagossomos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Apoptose , Lisossomos/metabolismo
12.
Med Sci (Paris) ; 40(3): 275-282, 2024 Mar.
Artigo em Francês | MEDLINE | ID: mdl-38520103

RESUMO

Cells can be reprogrammed into senescence to adapt to a variety of stresses, most often affecting the genome integrity. Senescent cells accumulate with age or upon various insults in almost all tissues, and contribute to the development of several age-associated pathologies. Studying the molecular pathways involved in senescence induction, maintenance, or escape is challenged by the heterogeneity in the level of commitment to senescence, and by the pollution of senescent cell populations by proliferating pre- or post-senescent cells. We coped with these difficulties by developing a protocol for sorting senescent cells by flow cytometry, based on three major senescence markers : the SA-ß-Galactosidase activity, the size of the cells, and their granularity reflecting the accumulation of aggregates, lysosomes, and altered mitochondria. We address the issues related to sorting senescent cells, the pitfalls to avoid, and propose solutions for sorting viable cells expressing senescent markers at different extents.


Title: Tri des cellules sénescentes par cytométrie en flux - Des spécificitéset des pièges à éviter. Abstract: La sénescence est un état d'adaptation des cellules au stress qui contribue au vieillissement et au développement de nombreuses maladies. Étudier les voies moléculaires modulant l'induction, le maintien ou l'échappement de la sénescence est compliqué par la contamination des populations de cellules sénescentes par des cellules proliférantes pré- ou post-sénescentes. Pour contourner cette difficulté, nous avons développé un protocole de tri par cytométrie en flux, fondé sur trois marqueurs majeurs de sénescence (l'activité SA-ß-galactosidase, la taille et la granularité des cellules), qui permet de trier des cellules sénescentes viables, à des degrés choisis d'engagement dans le phénotype.


Assuntos
Senescência Celular , Lisossomos , Humanos , Senescência Celular/genética , Citometria de Fluxo
13.
Cell Host Microbe ; 32(4): 466-478.e11, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38479395

RESUMO

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.


Assuntos
Citomegalovirus , Proteômica , Humanos , Citomegalovirus/fisiologia , Montagem de Vírus , Replicação Viral , Proteínas , Autofagia , Lisossomos , Concentração de Íons de Hidrogênio
14.
Mol Cell ; 84(7): 1354-1364.e9, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38447580

RESUMO

Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.


Assuntos
Glicoproteínas de Membrana , Lipofuscinoses Ceroides Neuronais , Camundongos , Animais , Criança , Humanos , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Lisossomos/metabolismo , Fosfolipases/metabolismo , Glicerofosfolipídeos/metabolismo , Fosfolipídeos/metabolismo
15.
Talanta ; 273: 125894, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38461644

RESUMO

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that can be used as a marker for the occurrence of oxidative stress in the organism. Lysosomes serve as intracellular digestive sites, and when the concentration of H2O2 in them is abnormal, lysosomal function is often impaired, leading to the development of diseases. Hydrogen sulfide (H2S) acts as a gaseous signaling molecule that scavenges H2O2 from cells and tissues, thereby maintaining the redox environment of the body. However, most of the reported hydrogen peroxide fluorescent probes so far can only detect H2O2, but cannot maintain the intracellular redox environment. In this paper, an H2O2 fluorescent probe LN-HOD with lysosomal targeting properties was designed and synthesized by combining the H2O2 recognition site with a naphthylamine fluorophore via a thiocarbamate moiety. The probe has the advantages of large Stokes shift (110 nm), high sensitivity and good H2S release capability. The probe LN-HOD can be used to detect H2O2 in cells, zebrafish and plant roots. In addition, LN-HOD detects changes in the concentration of H2O2 in plant roots when Arabidopsis is stressed by cadmium ion (Cd2+). And through its ability to release H2S, it can help to remove excess H2O2 and maintain the redox environment in cells, zebrafish and plant roots. The present work provides new ideas for the detection and assisted removal of H2O2, which contributes to the in-depth study of the cellular microenvironment in organisms.


Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Animais , Humanos , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Peixe-Zebra , Sulfeto de Hidrogênio/metabolismo , Oxirredução , Lisossomos/metabolismo , Células HeLa
16.
Ecotoxicol Environ Saf ; 274: 116195, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38479315

RESUMO

Fluoride is known to induce nephrotoxicity; however, the underlying mechanisms remain incompletely understood. Therefore, this study aims to explore the roles and mechanisms of lysosomal membrane permeabilization (LMP) and the GSDME/HMGB1 axis in fluoride-induced nephrotoxicity and the protective effects of rutin. Rutin, a naturally occurring flavonoid compound known for its antioxidative and anti-inflammatory properties, is primarily mediated by inhibiting oxidative stress and reducing proinflammatory markers. To that end, we established in vivo and in vitro models. In the in vivo study, rats were exposed to sodium fluoride (NaF) throughout pregnancy and up until 2 months after birth. In parallel, we employed in vitro models using HK-2 cells treated with NaF, n-acetyl-L-cysteine (NAC), or rutin. We assessed lysosomal permeability through immunofluorescence and analyzed relevant protein expression via western blotting. Our findings showed that NaF exposure increased ROS levels, resulting in enhanced LMP and increased cathepsin B (CTSB) and D (CTSD) expression. Furthermore, the exposure to NaF resulted in the upregulation of cleaved PARP1, cleaved caspase-3, GSDME-N, and HMGB1 expressions, indicating cell death and inflammation-induced renal damage. Rutin mitigates fluoride-induced nephrotoxicity by suppressing ROS-mediated LMP and the GSDME/HMGB1 axis, ultimately preventing fluoride-induced renal toxicity occurrence and development. In conclusion, our findings suggest that NaF induces renal damage through ROS-mediated activation of LMP and the GSDME/HMGB1 axis, leading to pyroptosis and inflammation. Rutin, a natural antioxidative and anti-inflammatory dietary supplement, offers a novel approach to prevent and treat fluoride-induced nephrotoxicity.


Assuntos
Fluoretos , Proteína HMGB1 , Nefropatias , Rutina , Animais , Ratos , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Caspase 3/metabolismo , Fluoretos/metabolismo , Fluoretos/toxicidade , Proteína HMGB1/efeitos dos fármacos , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Lisossomos/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Rutina/farmacologia , Fluoreto de Sódio/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/tratamento farmacológico , Gasderminas/efeitos dos fármacos , Gasderminas/metabolismo
17.
Eur J Med Chem ; 269: 116329, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38508117

RESUMO

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.


Assuntos
Catepsina B , Catepsina B/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Lisossomos/química , Lisossomos/metabolismo
18.
Int J Mol Sci ; 25(6)2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38542221

RESUMO

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.


Assuntos
Canabinoides , Infecções por HIV , HIV-1 , Humanos , Feminino , NF-kappa B/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Macrófagos/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Estresse Oxidativo , Exocitose , Lisossomos/metabolismo
19.
J Ethnopharmacol ; 328: 118076, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38521431

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: QiXian Granule (QXG) is an integrated traditional Chinese medicine formula used to treat postmenopausal atherosclerotic (AS) cardiovascular diseases. The previous studies have found that QXG inhibited isoproterenol (ISO)-induced myocardial remodeling. And its active ingredient, Icraiin, can inhibit ferroptosis by promoting oxidized low-density lipoprotein (xo-LDL)-induced vascular endothelial cell injury and autophagy in atherosclerotic mice. Another active ingredient, Salvianolic Acid B, can suppress ferroptosis and apoptosis during myocardial ischemia/reperfusion injury by reducing ubiquitin-proteasome degradation of Glutathione Peroxidase 4 (GPX4) and down-regulating the reactive oxygen species (ROS)- c-Jun N-terminal kinases (JNK)/mitogen-activated protein kinase (MAPK) pathway. AIM OF THE STUDY: The objective of this research was to assess the possible impact of QXG on atherosclerosis in postmenopausal individuals and investigate its underlying mechanisms. MATERIALS AND METHODS: Female ApoE-/- mice underwent ovariectomy and were subjected to a high-fat diet (HFD) to establish a postmenopausal atherosclerosis model. The therapeutic effects of QXG were observed in vivo and in vitro through intraperitoneal injection of erastin, G-protein Coupled Estrogen Receptor (GPER) inhibitor (G15), and silent Mucolipin Transient Receptor Potential Channel 1 (TRPML1) adenovirus injection via tail vein. UPLC-MS and molecular docking techniques identified and evaluated major QXG components, contributing to the investigation of QXG's anti-postmenopausal atherosclerotic effects. RESULTS: QXG increased serum Estradiol levels, decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, which indicated QXG had estrogen-like effects in Ovx/ApoE-/- mice. Furthermore, QXG demonstrated the potential to impede the progression of AS in Ovx/ApoE-/- mice, as evidenced by reductions in serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels. Additionally, QXG inhibited ferroptosis in Ovx/ApoE-/- mice. Notably, UPLC-MS analysis identified a total of 106 active components in QXG. The results of molecular docking analysis demonstrated that Epmedin B, Astragaloside II, and Orientin exhibit strong binding affinity towards TRPML1. QXG alleviates the progression of atherosclerosis by activating TRPML1 through the GPER pathway or directly activating TRPML1, thereby inhibiting GPX4 and ferritin heavy chain (FTH1)-mediated iron pendant disease. In vitro, QXG-treated serum suppressed proliferation, migration, and ox-LDL-induced MMP and ROS elevation in HAECs. CONCLUSION: QXG inhibited GPX4 and FTH1-mediated ferroptosis in vascular endothelial cells through up-regulating GPER/TRPML1 signaling, providing a potential therapeutic option for postmenopausal females seeking a safe and effective medication to prevent atherosclerosis. The study highlights QXG's estrogenic properties and its promising role in combating postmenopausal atherosclerosis.


Assuntos
Aterosclerose , Medicamentos de Ervas Chinesas , Ferroptose , Feminino , Animais , Camundongos , Células Endoteliais , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Pós-Menopausa , Cromatografia Líquida , Simulação de Acoplamento Molecular , Espectrometria de Massas em Tandem , Aterosclerose/tratamento farmacológico , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , LDL-Colesterol/metabolismo , Estrogênios/metabolismo , Apolipoproteínas E , Lisossomos/metabolismo
20.
Nat Commun ; 15(1): 2779, 2024 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-38555350

RESUMO

Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.


Assuntos
Resistência à Insulina , Obesidade , Masculino , Camundongos , Animais , Obesidade/complicações , Obesidade/genética , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Dieta Hiperlipídica/efeitos adversos , Lisossomos/metabolismo , Lipídeos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL
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