Alzheimer’s disease is a multi-amyloidosis disease characterized by amyloid-βdeposits in brain blood vessels,microaneurysms,and senile plaques.How amyloid-βdeposition affects axon pathology has not been examined ex...Alzheimer’s disease is a multi-amyloidosis disease characterized by amyloid-βdeposits in brain blood vessels,microaneurysms,and senile plaques.How amyloid-βdeposition affects axon pathology has not been examined extensively.We used immunohistochemistry and immunofluorescence staining to analyze the forebrain tissue slices of Alzheimer’s disease patients.Widespread axonal amyloidosis with distinctive axonal enlargement was observed in patients with Alzheimer’s disease.On average,amyloid-β-positive axon diameters in Alzheimer’s disease brains were 1.72 times those of control brain axons.Furthermore,axonal amyloidosis was associated with microtubule-associated protein 2 reduction,tau phosphorylation,lysosome destabilization,and several blood-related markers,such as apolipoprotein E,alpha-hemoglobin,glycosylated hemoglobin type A1C,and hemin.Lysosome destabilization in Alzheimer’s disease was also clearly identified in the neuronal soma,where it was associated with the co-expression of amyloid-β,Cathepsin D,alpha-hemoglobin,actin alpha 2,and collagen type IV.This suggests that exogenous hemorrhagic protein intake influences neural lysosome stability.Additionally,the data showed that amyloid-β-containing lysosomes were 2.23 times larger than control lysosomes.Furthermore,under rare conditions,axonal breakages were observed,which likely resulted in Wallerian degeneration.In summary,axonal enlargement associated with amyloidosis,micro-bleeding,and lysosome destabilization is a major defect in patients with Alzheimer’s disease.This finding suggests that,in addition to the well-documented neural soma and synaptic damage,axonal damage is a key component of neuronal defects in Alzheimer’s disease.展开更多
Ovarian cancer(OC),a common malignancy of the female reproductive system,has the highest mortality rate among gynecological cancers.A distinguishing feature of OC cells(OCCs)is their reduced autophagic flux compared w...Ovarian cancer(OC),a common malignancy of the female reproductive system,has the highest mortality rate among gynecological cancers.A distinguishing feature of OC cells(OCCs)is their reduced autophagic flux compared with normal cells.This phenomenon indicates that excessive autophagy activation or impaired autophagosome–lysosome fusion may lead to OCC death.This study investigated the anti-OC effects of dihydrotanshinone I(DHT),a tanshinone compound from Salvia miltiorrhiza.Proteomic analysis suggested that DHT suppressed OC growth via the autophagy–lysosome pathway,with sortilin 1(SORT1)identified as a critical target.In vitro,DHT promoted autophagosome formation mediated by microtubule-associated protein 1 light chain 3-II(LC3-II),while inhibiting autophagosome–lysosome fusion.The results of an orthotopic OC model corroborated these findings,showing that DHT induced autophagic cell death(ACD)and suppressed SORT1 expression in tumors.Further RNA interference experiments confirmed that SORT1 depletion caused autophagosomes to accumulate in OCCs.Notably,we found that SORT1 interacted with autophagy-related gene(ATG)-encoded proteins ATG5 and ATG16L1,and that depleting SORT1 increased the levels of these proteins.Co-immunoprecipitation,ubiquitination,and cellular thermal shift assay analyses revealed that DHT directly targeted and promoted ubiquitin-dependent degradation of SORT1.By degrading SORT1,ATG5 and ATG16L1 were released,which enhanced autophagosome formation and disrupted the autophagic flux.These findings identified DHT as a novel autophagosome inducer that induced ACD by targeting SORT1,making it a promising therapeutic candidate for OC.展开更多
Objective To identify prognostic genes associated with lysosome-dependent cell death(LDCD)in patients with gastric cancer(GC).Methods Differentially expressed genes(DEGs)were identified using The Cancer Genome Atlas-S...Objective To identify prognostic genes associated with lysosome-dependent cell death(LDCD)in patients with gastric cancer(GC).Methods Differentially expressed genes(DEGs)were identified using The Cancer Genome Atlas-Stomach Adenocarcinoma.Weighted gene co-expression network analysis was performed to identify the key module genes associated with LDCD score.Candidate genes were identified by DEGs and key module genes.Univariate Cox regression analysis,and least absolute shrinkage and selection operator regression and multivariate Cox regression analyses were performed for the selection of prognostic genes,and risk module was established.Subsequently,key cells were identified in the single-cell dataset(GSE183904),and prognostic gene expression was analyzed.Cell proliferation and migration were assessed using the Cell Counting Kit-8 assay and the wound healing assay.Results A total of 4,465 DEGs,95 candidate genes,and 4 prognostic genes,including C19orf59,BATF2,TNFAIP2,and TNFSF18,were identified in the analysis.Receiver operating characteristic curves indicated the excellent predictive power of the risk model.Three key cell types(B cells,chief cells,and endothelial/pericyte cells)were identified in the GSE183904 dataset.C19orf59 and TNFAIP2 exhibited predominant expression in macrophage species,whereas TNFAIP2 evolved over time in endothelial/pericyte cells and chief cells.Functional experiments confirmed that interfering with C19orf59 inhibited proliferation and migration in GC cells.Conclusion C19orf59,BATF2,TNFAIP2,and TNFSF18 are prognostic genes associated with LDCD in GC.Furthermore,the risk model established in this study showed robust predictive power.展开更多
Therapeutic progress in neurodegenerative conditions such as Parkinson’s disease has been hampered by a lack of detailed knowledge of its molecular etiology.The advancements in genetics and genomics have provided fun...Therapeutic progress in neurodegenerative conditions such as Parkinson’s disease has been hampered by a lack of detailed knowledge of its molecular etiology.The advancements in genetics and genomics have provided fundamental insights into specific protein players and the cellular processes involved in the onset of disease.In this respect,the autophagy-lysosome system has emerged in recent years as a strong point of convergence for genetics,genomics,and pathologic indications,spanning both familial and idiopathic Parkinson’s disease.Most,if not all,genes linked to familial disease are involved,in a regulatory capacity,in lysosome function(e.g.,LRRK2,alpha-synuclein,VPS35,Parkin,and PINK1).Moreover,the majority of genomic loci associated with increased risk of idiopathic Parkinson’s cluster in lysosome biology and regulation(GBA as the prime example).Lastly,neuropathologic evidence showed alterations in lysosome markers in autoptic material that,coupled to the alpha-synuclein proteinopathy that defines the disease,strongly indicate an alteration in functionality.In this Brief Review article,I present a personal perspective on the molecular and cellular involvement of lysosome biology in Parkinson’s pathogenesis,aiming at a larger vision on the events underlying the onset of the disease.The attempts at targeting autophagy for therapeutic purposes in Parkinson’s have been mostly aimed at“indiscriminately”enhancing its activity to promote the degradation and elimination of aggregate protein accumulations,such as alpha-synuclein Lewy bodies.However,this approach is based on the assumption that protein pathology is the root cause of disease,while pre-pathology and pre-degeneration dysfunctions have been largely observed in clinical and pre-clinical settings.In addition,it has been reported that unspecific boosting of autophagy can be detrimental.Thus,it is important to understand the mechanisms of specific autophagy forms and,even more,the adjustment of specific lysosome functionalities.Indeed,lysosomes exert fine signaling capacities in addition to their catabolic roles and might participate in the regulation of neuronal and glial cell functions.Here,I discuss hypotheses on these possible mechanisms,their links with etiologic and risk factors for Parkinson’s disease,and how they could be targeted for disease-modifying purposes.展开更多
The prevalence of cardiovascular diseases(CVDs)has increased markedly as the world population has aged.Long non-coding RNAs(lncRNAs)have been reported as novel regulators in diverse pathophysiological conditions.Here,...The prevalence of cardiovascular diseases(CVDs)has increased markedly as the world population has aged.Long non-coding RNAs(lncRNAs)have been reported as novel regulators in diverse pathophysiological conditions.Here,we performed RNA sequencing(RNA-seq)and observed that the lncRNA Zeb1os1(zinc finger E-box binding homeobox 1,opposite strand 1),which is known as ZEB1-AS1(zinc finger E-box binding homeobox 1 antisense 1)in humans,was upregulated in the aged mice hearts,senescent cardiomyocytes,and human blood from elderly individuals.The human blood ZEB1-AS1 level was positively relevant to human age but negatively relevant to peak E to peak A(E/A).Silencing Zeb1os1 ameliorated diastolic dysfunction and cardiac senescence in aged mice.On the other hand,Zeb1os1 overexpression triggered cardiac dysfunction resembling that observed in aged mice.Mechanistically,we provide compelling evidence that Zeb1os1 interacts with the transient receptor potential mucolipin 1(TRPML1)for ubiquitination(UB)-mediated degradation.This process inhibits lysosomal Ca^(2+)efflux,impairing lysosome function.In addition,the functional domain of Zeb1os1,which contains the key nucleotides responsible for the pro-senescence property of full-length Zeb1os1 in cardiomyocytes.Together,these data suggest that Zeb1os1 is a potential target for ameliorating lysosomal dysfunction and aging-related cardiac impairment.展开更多
Lysosomes are discrete organelles that act as recycling centers for extracellular and intracellular materials,playing a pivotal role in maintaining cellular homeostasis.Their acidic environment,maintained by numerous ...Lysosomes are discrete organelles that act as recycling centers for extracellular and intracellular materials,playing a pivotal role in maintaining cellular homeostasis.Their acidic environment,maintained by numerous hydrolytic enzymes,facilitates substrate degradation.Dysfunction in lysosomal processes can lead to abnormal substrate degradation,significantly impacting cellular homeostasis.High energy-demanding cells,such as post-mitotic neurons,are especially vulnerable to these changes,often resulting in neurological diseases.Autophagy,a conserved catabolic process,requires extensive lysosomal utilization.It plays a key role in removing unnecessary intracellular components,ensuring cellular homeostasis,and promoting cell survival during stress conditions such as starvation,infection,or cellular damage.展开更多
Millions of people are suffering from Alzheimer’s disease globally,but there is still no effective treatment for this neurodegenerative disease.Thus,novel therapeutic approaches for Alzheimer’s disease are needed,wh...Millions of people are suffering from Alzheimer’s disease globally,but there is still no effective treatment for this neurodegenerative disease.Thus,novel therapeutic approaches for Alzheimer’s disease are needed,which requires further evaluation of the regulato ry mechanisms of protein aggregate degradation.Lysosomes are crucial degradative organelles that maintain cellular homeostasis.Transcription factor EB-mediated lysosome biogenesis enhances autolysosomedependent degradation,which subsequently alleviates neurodege nerative diseases,including Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease.In this review,we start by describing the key features of lysosomes,including their roles in nutrient sensing and degradation,and their functional impairments in different neurodegenerative diseases.We also explain the mechanisms—especially the post-translational modifications—which impact transcription factor EB and regulate lysosome biogenesis.Next,we discuss strategies for promoting the degradation of toxic protein aggregates.We describe Proteolysis-Ta rgeting Chimera and related technologies for the targeted degradation of specific proteins.We also introduce a group of LYsosome-Enhancing Compounds,which promote transcription factor EB-mediated lysosome biogenesis and improve learning,memory,and cognitive function in APP-PSEN1 mice.In summary,this review highlights the key aspects of lysosome biology,the mechanisms of transcription factor EB activation and lysosome biogenesis,and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.展开更多
Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ...Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.展开更多
Mitochondria and lysosomes are essential cellular organelles in most eukaryotic cells by playing the physiological roles to support the normal functions of cells, as well as the life of the whole body. To date,small-m...Mitochondria and lysosomes are essential cellular organelles in most eukaryotic cells by playing the physiological roles to support the normal functions of cells, as well as the life of the whole body. To date,small-molecule fluorescent probes have been considered as one of the vital tools for monitoring and visualizing multiple biological analytes. This review summarized the recent advances in small-molecule two-photon fluorescent probes for metal ions, reactive oxygen species（ROS） and reactive sulfur species（RSS）, and changes inside micro-environment（e.g., p H, viscosity and polarity） in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.展开更多
The altered lysosomal function can induce drug redistribution which leads to drug resistance and poor prognosis for cancer patients.V-ATPase,an ATP-driven proton pump positioned at lysosomal surfaces,is responsible fo...The altered lysosomal function can induce drug redistribution which leads to drug resistance and poor prognosis for cancer patients.V-ATPase,an ATP-driven proton pump positioned at lysosomal surfaces,is responsible for maintaining the stability of lysosome.Herein,we reported that the potassium voltage-gated channel subfamily J member 15(KCNJ15)protein,which may bind to V-ATPase,can regulate the function of lysosome.The deficiency of KCNJ15 protein in breast cancer cells led to drug aggregation as well as reduction of drug efficacy.The application of the V-ATPase inhibitor could inhibit the binding between KCNJ15 and V-ATPase,contributing to the amelioration of drug resistance.Clinical data analysis revealed that KCNJ15 deficiency was associated with higher histological grading,advanced stages,more metastases of lymph nodes,and shorter disease free survival of patients with breast cancer.KCNJ15 expression level is positively correlated with a high response rate after receiving neoadjuvant chemotherapy.Moreover,we revealed that the small molecule drug CMA/BAF can reverse drug resistance by disrupting the interaction between KCNJ15 and lysosomes.In conclusion,KCNJ15 could be identified as an underlying indicator for drug resistance and survival of breast cancer,which might guide the choice of therapeutic strategies.展开更多
Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, ...Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, rapid amplification of cDNA ends （RACE） and reverse transcription polymerase chain reaction （RT-PCR）. Previous study showed that the novel gene played an important role in the occurrence, development, migration and prognosis of tumors. Pancreatic cancer is an aggressive malignancy with the majority of patients dying within one year after diagnosis. This study tries to find out the relationship between lysosome associated protein transmembrane 4 beta gene polymorphism and the susceptibility of pancreatic cancer. Methods： A case-control study was conducted in China, including 58 pancreatic cancer cases and 156 healthy controls. Human genomic DNA was used as the template, polymerase chain reaction （PCR） was used to detect the distribution of LAPTM4B genotype. Analyses Odds ratio （OR） and corresponding 95% confidence interval （95%CI） with logistic regression were performed. Results： Two alleles of LAPTM4B generated three kinds of genotypes in population, ＊1/1, ＊1/2, and ＊2/2. The genotype frequency of ＊1/1, ＊1/2 and ＊2/2 in the pancreatic cancer group were 41.4%, 44.8% and 13.8% respectively, which were not significantly different from those of healthy group （47.4%, 42.9%, 9.6%） （P=0.773, P=0.291）. Also the ＊2 allele frequency of LAPTM4B among pancreatic cancer had no significantly difference with the controls （P=0.354）. When compared to the ＊1 allele, the people with ＊2 allele had no increased risk of pancreatic cancer. Conclusion： The gene polymorphism of LAPTM4B may not influence the susceptibility of pancreatic cancer.展开更多
OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer,but the anticancer effect of chloroquine alone is limited.Since we found that the flavonoid kaempferol effectively sensitizes...OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer,but the anticancer effect of chloroquine alone is limited.Since we found that the flavonoid kaempferol effectively sensitizes glioma cells to chloroquine-mediated cell death,we investigated the underlying mechanisms of glioma cell death induced by the combination of kaempferol and chloroquine.METHODS To examine the effect of kaempferol and/or chloroquine on various glioma cells,cell viability assay using calcein-AM and EthD-1was performed.The changes in the lysosomal structures following treatment with kaempferol and/or chloroquine were observed by electron microscopy and fluorescence microscopy using acridine orange or Lyso-tracker Red.The changes in cathepsin D proteins were analyzed by Western blotting,immunocytochemistry,and fluorescence microscopy using BODIPY FL-pepstatin.RESULTS Treatment with subtoxic doses of chloroquine,when combined with kaempferol,effectively induced cell death in various glioma cells,but not in normal astrocytes.While kaempferol treatment increased the numbers of lysosome,chloroquine treatment increased lysosomal masses.Combined treatment with kaempferol and chloroquine induced the expansion and subsequent rupture of lysosomes,leading to the spillage of the lysosomal contents into the cytosol.We found that while kaemfperol treatment increased the active mature forms of cathepsin D,chloroquine treatment completely blocked the processing of cathepsin D.The processing of cathepsin D was also blocked by the combined treatment,but the activity of cathepsin D,which was released from the lysosomes,was restored.The cell death induced by kaempferol and chloroquine in U251 MG cells was accompanied by mitochondrial dysfunction,ER stress,and DNA damage.CONCLUSION Disruption of lysosomal membrane integrity and a resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by chloroquine plus kaempferol.展开更多
OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients wer...OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.展开更多
Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PC...Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.展开更多
Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer lea...Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)—susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core—carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.展开更多
The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosisinducing activity in various cells of different origins. Here, we present evidence that the underlying molecular...The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosisinducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I （PCD I, apoptosis） and PCD II （autophagy）-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atgl2-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class Ⅲ inhibitor, 3-methyladenine （3-MA）, and by the vacuole H＋-ATPase inhibitor, bafilomycin-A1 （Baf-A1）. S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ATM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species （ROS） generation, and partially pro- tected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated ceils. In addition, either ATM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.展开更多
Carbon-based fluorescent nanomaterials have gained much attention in recent years.In this work,greenphotoluminescent carbon nanodots(CNDs;also termed carbon dots,CDs)with amine termination were synthesized via the hyd...Carbon-based fluorescent nanomaterials have gained much attention in recent years.In this work,greenphotoluminescent carbon nanodots(CNDs;also termed carbon dots,CDs)with amine termination were synthesized via the hydrothermal treatment of amine-containing spermine and rose bengal(RB)molecules.The CNDs have an ultrasmall size of~2.2 nm and present bright photoluminescence with a high quantum yield of~80%which is possibly attributed to the loss of halogen atoms(Cl and I)during the hydrothermal reaction.Different from most CNDs which have multicolor fluorescence emission,the asprepared CNDs possess excitation-independent emission property,which can avoid fluorescence overlap with other fluorescent dyes.Moreover,the weakly basic amine-terminated surface endows the CNDs with the acidotropic effect.As a result,the CNDs can accumulate in the acidic lysosomes after cellular internalization and can serve as a favorable agent for lysosome imaging.Besides,the CNDs have a negligible impact on the lysosomal morphology even after 48 h incubation and exhibit excellent biocompatibility in the used cell models.展开更多
Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the s...Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the study,a novel HIF-1αASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA.The physicochemical properties of nanocomposite were characterized using TEM,FTIR,DLS and zeta potentials.The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm,and according to the agarose gel retardation assay,the loading amount of ASODN accounted for 166.7 mg/g.The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells.The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL.The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite.In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit m RNA expression and HIF-1αprotein translation at different levels.In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue.In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%.These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.展开更多
Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organellespecific pH...Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organellespecific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe(BLT) with lysosomes localization capability was developed.The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable p Kavalue has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced(APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.展开更多
Targeted protein degradation(TPD)has transformed drug discovery by eliminating disease-causing proteins rather than merely inhibiting their activity.Proteolysis-targeting chimeras(PROTACs)have significantly advanced t...Targeted protein degradation(TPD)has transformed drug discovery by eliminating disease-causing proteins rather than merely inhibiting their activity.Proteolysis-targeting chimeras(PROTACs)have significantly advanced this field by using bifunctional small molecules to recruit E3 ubiquitin ligases to degrade proteins of interest.However,PROTACs,relying on the ubiquitin-proteasome system,predominantly target cytosolic and nuclear proteins,but they struggle to degrade membrane or extracellular proteins.To address this gap,scientists have developed lysosome-targeting chimeras(LYTACs),which consist of an antibody or peptide binding the target protein,linked to a ligand that binds a cellsurface lysosomal trafficking receptor.By bridging a target protein on the cell surface to a lysosome-shuttling receptor,LYTACs are internalized into the cell and delivered to lysosomes,where acidic enzymes degrade various membrane proteins.In essence,LYTACs broaden the druggable proteome,allowing researchers to modulate“undruggable”targets that PROTACs and traditional inhibitors cannot touch.展开更多
基金supported by the National Natural Science Foundation of China,No.81472235(to HF)the Shanghai Jiao Tong University Medical and Engineering Project,Nos.YG2021QN53(to HF),YG2017MS71(to HF)+1 种基金the International Cooperation Project of the National Natural Science Foundation of China,No.82020108017(to DC)the Innovation Group Project of the National Natural Science Foundation of China,No.81921002(to DC).
文摘Alzheimer’s disease is a multi-amyloidosis disease characterized by amyloid-βdeposits in brain blood vessels,microaneurysms,and senile plaques.How amyloid-βdeposition affects axon pathology has not been examined extensively.We used immunohistochemistry and immunofluorescence staining to analyze the forebrain tissue slices of Alzheimer’s disease patients.Widespread axonal amyloidosis with distinctive axonal enlargement was observed in patients with Alzheimer’s disease.On average,amyloid-β-positive axon diameters in Alzheimer’s disease brains were 1.72 times those of control brain axons.Furthermore,axonal amyloidosis was associated with microtubule-associated protein 2 reduction,tau phosphorylation,lysosome destabilization,and several blood-related markers,such as apolipoprotein E,alpha-hemoglobin,glycosylated hemoglobin type A1C,and hemin.Lysosome destabilization in Alzheimer’s disease was also clearly identified in the neuronal soma,where it was associated with the co-expression of amyloid-β,Cathepsin D,alpha-hemoglobin,actin alpha 2,and collagen type IV.This suggests that exogenous hemorrhagic protein intake influences neural lysosome stability.Additionally,the data showed that amyloid-β-containing lysosomes were 2.23 times larger than control lysosomes.Furthermore,under rare conditions,axonal breakages were observed,which likely resulted in Wallerian degeneration.In summary,axonal enlargement associated with amyloidosis,micro-bleeding,and lysosome destabilization is a major defect in patients with Alzheimer’s disease.This finding suggests that,in addition to the well-documented neural soma and synaptic damage,axonal damage is a key component of neuronal defects in Alzheimer’s disease.
基金supported by the National Key Research and Development Program of China(2023YFC3503900)the National Natural Science Foundation of China(82305001)+3 种基金the Zhejiang Provincial Natural Science Foundation of China(LQ24H280011)the Science Research Fund of Administration of Traditional Chinese Medicine of Zhejiang Province(2023ZR014)the National Young Qihuang Scholars Training Programthe Research Project of Zhejiang Chinese Medical University(2022RCZXZK18,2023JKZKTS17)。
文摘Ovarian cancer(OC),a common malignancy of the female reproductive system,has the highest mortality rate among gynecological cancers.A distinguishing feature of OC cells(OCCs)is their reduced autophagic flux compared with normal cells.This phenomenon indicates that excessive autophagy activation or impaired autophagosome–lysosome fusion may lead to OCC death.This study investigated the anti-OC effects of dihydrotanshinone I(DHT),a tanshinone compound from Salvia miltiorrhiza.Proteomic analysis suggested that DHT suppressed OC growth via the autophagy–lysosome pathway,with sortilin 1(SORT1)identified as a critical target.In vitro,DHT promoted autophagosome formation mediated by microtubule-associated protein 1 light chain 3-II(LC3-II),while inhibiting autophagosome–lysosome fusion.The results of an orthotopic OC model corroborated these findings,showing that DHT induced autophagic cell death(ACD)and suppressed SORT1 expression in tumors.Further RNA interference experiments confirmed that SORT1 depletion caused autophagosomes to accumulate in OCCs.Notably,we found that SORT1 interacted with autophagy-related gene(ATG)-encoded proteins ATG5 and ATG16L1,and that depleting SORT1 increased the levels of these proteins.Co-immunoprecipitation,ubiquitination,and cellular thermal shift assay analyses revealed that DHT directly targeted and promoted ubiquitin-dependent degradation of SORT1.By degrading SORT1,ATG5 and ATG16L1 were released,which enhanced autophagosome formation and disrupted the autophagic flux.These findings identified DHT as a novel autophagosome inducer that induced ACD by targeting SORT1,making it a promising therapeutic candidate for OC.
基金supported by Hainan Provincial Natural Science Foundation of China(No.820CXTD438)National Natural Science Foundation of China(No.82160634, No.81773495)。
文摘Objective To identify prognostic genes associated with lysosome-dependent cell death(LDCD)in patients with gastric cancer(GC).Methods Differentially expressed genes(DEGs)were identified using The Cancer Genome Atlas-Stomach Adenocarcinoma.Weighted gene co-expression network analysis was performed to identify the key module genes associated with LDCD score.Candidate genes were identified by DEGs and key module genes.Univariate Cox regression analysis,and least absolute shrinkage and selection operator regression and multivariate Cox regression analyses were performed for the selection of prognostic genes,and risk module was established.Subsequently,key cells were identified in the single-cell dataset(GSE183904),and prognostic gene expression was analyzed.Cell proliferation and migration were assessed using the Cell Counting Kit-8 assay and the wound healing assay.Results A total of 4,465 DEGs,95 candidate genes,and 4 prognostic genes,including C19orf59,BATF2,TNFAIP2,and TNFSF18,were identified in the analysis.Receiver operating characteristic curves indicated the excellent predictive power of the risk model.Three key cell types(B cells,chief cells,and endothelial/pericyte cells)were identified in the GSE183904 dataset.C19orf59 and TNFAIP2 exhibited predominant expression in macrophage species,whereas TNFAIP2 evolved over time in endothelial/pericyte cells and chief cells.Functional experiments confirmed that interfering with C19orf59 inhibited proliferation and migration in GC cells.Conclusion C19orf59,BATF2,TNFAIP2,and TNFSF18 are prognostic genes associated with LDCD in GC.Furthermore,the risk model established in this study showed robust predictive power.
基金supported by grants from Parkinson Canada,The Weston Brain Foundation and the Euregio Science Fund(to MV).
文摘Therapeutic progress in neurodegenerative conditions such as Parkinson’s disease has been hampered by a lack of detailed knowledge of its molecular etiology.The advancements in genetics and genomics have provided fundamental insights into specific protein players and the cellular processes involved in the onset of disease.In this respect,the autophagy-lysosome system has emerged in recent years as a strong point of convergence for genetics,genomics,and pathologic indications,spanning both familial and idiopathic Parkinson’s disease.Most,if not all,genes linked to familial disease are involved,in a regulatory capacity,in lysosome function(e.g.,LRRK2,alpha-synuclein,VPS35,Parkin,and PINK1).Moreover,the majority of genomic loci associated with increased risk of idiopathic Parkinson’s cluster in lysosome biology and regulation(GBA as the prime example).Lastly,neuropathologic evidence showed alterations in lysosome markers in autoptic material that,coupled to the alpha-synuclein proteinopathy that defines the disease,strongly indicate an alteration in functionality.In this Brief Review article,I present a personal perspective on the molecular and cellular involvement of lysosome biology in Parkinson’s pathogenesis,aiming at a larger vision on the events underlying the onset of the disease.The attempts at targeting autophagy for therapeutic purposes in Parkinson’s have been mostly aimed at“indiscriminately”enhancing its activity to promote the degradation and elimination of aggregate protein accumulations,such as alpha-synuclein Lewy bodies.However,this approach is based on the assumption that protein pathology is the root cause of disease,while pre-pathology and pre-degeneration dysfunctions have been largely observed in clinical and pre-clinical settings.In addition,it has been reported that unspecific boosting of autophagy can be detrimental.Thus,it is important to understand the mechanisms of specific autophagy forms and,even more,the adjustment of specific lysosome functionalities.Indeed,lysosomes exert fine signaling capacities in addition to their catabolic roles and might participate in the regulation of neuronal and glial cell functions.Here,I discuss hypotheses on these possible mechanisms,their links with etiologic and risk factors for Parkinson’s disease,and how they could be targeted for disease-modifying purposes.
基金funded by the National Natural Science Foundation of China(82273919,82270396,and U21A20339)the China Postdoctoral Science Foundation(2023T160176)。
文摘The prevalence of cardiovascular diseases(CVDs)has increased markedly as the world population has aged.Long non-coding RNAs(lncRNAs)have been reported as novel regulators in diverse pathophysiological conditions.Here,we performed RNA sequencing(RNA-seq)and observed that the lncRNA Zeb1os1(zinc finger E-box binding homeobox 1,opposite strand 1),which is known as ZEB1-AS1(zinc finger E-box binding homeobox 1 antisense 1)in humans,was upregulated in the aged mice hearts,senescent cardiomyocytes,and human blood from elderly individuals.The human blood ZEB1-AS1 level was positively relevant to human age but negatively relevant to peak E to peak A(E/A).Silencing Zeb1os1 ameliorated diastolic dysfunction and cardiac senescence in aged mice.On the other hand,Zeb1os1 overexpression triggered cardiac dysfunction resembling that observed in aged mice.Mechanistically,we provide compelling evidence that Zeb1os1 interacts with the transient receptor potential mucolipin 1(TRPML1)for ubiquitination(UB)-mediated degradation.This process inhibits lysosomal Ca^(2+)efflux,impairing lysosome function.In addition,the functional domain of Zeb1os1,which contains the key nucleotides responsible for the pro-senescence property of full-length Zeb1os1 in cardiomyocytes.Together,these data suggest that Zeb1os1 is a potential target for ameliorating lysosomal dysfunction and aging-related cardiac impairment.
文摘Lysosomes are discrete organelles that act as recycling centers for extracellular and intracellular materials,playing a pivotal role in maintaining cellular homeostasis.Their acidic environment,maintained by numerous hydrolytic enzymes,facilitates substrate degradation.Dysfunction in lysosomal processes can lead to abnormal substrate degradation,significantly impacting cellular homeostasis.High energy-demanding cells,such as post-mitotic neurons,are especially vulnerable to these changes,often resulting in neurological diseases.Autophagy,a conserved catabolic process,requires extensive lysosomal utilization.It plays a key role in removing unnecessary intracellular components,ensuring cellular homeostasis,and promoting cell survival during stress conditions such as starvation,infection,or cellular damage.
基金STI2030-Major Projects,No.2022ZD0213000the National Natural Science Foundation of China,Nos.92057103 and 31872820+1 种基金Shanghai Basic Research Program,No.18ZR1 404000State Key Laboratory of Drug Research,No.SIMM2004KF-09 (all to YL)。
文摘Millions of people are suffering from Alzheimer’s disease globally,but there is still no effective treatment for this neurodegenerative disease.Thus,novel therapeutic approaches for Alzheimer’s disease are needed,which requires further evaluation of the regulato ry mechanisms of protein aggregate degradation.Lysosomes are crucial degradative organelles that maintain cellular homeostasis.Transcription factor EB-mediated lysosome biogenesis enhances autolysosomedependent degradation,which subsequently alleviates neurodege nerative diseases,including Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease.In this review,we start by describing the key features of lysosomes,including their roles in nutrient sensing and degradation,and their functional impairments in different neurodegenerative diseases.We also explain the mechanisms—especially the post-translational modifications—which impact transcription factor EB and regulate lysosome biogenesis.Next,we discuss strategies for promoting the degradation of toxic protein aggregates.We describe Proteolysis-Ta rgeting Chimera and related technologies for the targeted degradation of specific proteins.We also introduce a group of LYsosome-Enhancing Compounds,which promote transcription factor EB-mediated lysosome biogenesis and improve learning,memory,and cognitive function in APP-PSEN1 mice.In summary,this review highlights the key aspects of lysosome biology,the mechanisms of transcription factor EB activation and lysosome biogenesis,and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.
基金supported by the National Natural Science Foundation of China,No.31670832,31470807,31270872a grant from the National Key Research and Development Program of China,No.2016YFA0500301a grant from the State Key Laboratory of Protein and Plant Gene Research,College of Life Sciences,Peking University,China
文摘Previous studies have shown that sirtuin 1（SIRT1） reduces the production of neuronal amyloid beta（Aβ） and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer＇s disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.
基金supported by the National Natural Science Foundation of China (Nos. 21778001, 21372005)the Anhui Provincial Natural Science Foundation (No. 1608085MB39)+1 种基金the Natural Science Foundation of Education Department of Anhui Province (No. KJ2015A047)the 211 Project of Anhui University
文摘Mitochondria and lysosomes are essential cellular organelles in most eukaryotic cells by playing the physiological roles to support the normal functions of cells, as well as the life of the whole body. To date,small-molecule fluorescent probes have been considered as one of the vital tools for monitoring and visualizing multiple biological analytes. This review summarized the recent advances in small-molecule two-photon fluorescent probes for metal ions, reactive oxygen species（ROS） and reactive sulfur species（RSS）, and changes inside micro-environment（e.g., p H, viscosity and polarity） in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.
基金supported by the National Natural Science Foundation of China(#81872159,#81902607,and#81874301)the Liaoning Colleges Innovative Talent Support Program(#Cancer Stem Cell Origin and Biology Behavior)+2 种基金the Major Project Construction Foundation of China Medical University(#2017ZDZX05)the Outstanding Scientific Fund of Shengjing Hospital(#201803)the Outstanding Young Scholars of Liaoning Province(#2019-YQ-10).
文摘The altered lysosomal function can induce drug redistribution which leads to drug resistance and poor prognosis for cancer patients.V-ATPase,an ATP-driven proton pump positioned at lysosomal surfaces,is responsible for maintaining the stability of lysosome.Herein,we reported that the potassium voltage-gated channel subfamily J member 15(KCNJ15)protein,which may bind to V-ATPase,can regulate the function of lysosome.The deficiency of KCNJ15 protein in breast cancer cells led to drug aggregation as well as reduction of drug efficacy.The application of the V-ATPase inhibitor could inhibit the binding between KCNJ15 and V-ATPase,contributing to the amelioration of drug resistance.Clinical data analysis revealed that KCNJ15 deficiency was associated with higher histological grading,advanced stages,more metastases of lymph nodes,and shorter disease free survival of patients with breast cancer.KCNJ15 expression level is positively correlated with a high response rate after receiving neoadjuvant chemotherapy.Moreover,we revealed that the small molecule drug CMA/BAF can reverse drug resistance by disrupting the interaction between KCNJ15 and lysosomes.In conclusion,KCNJ15 could be identified as an underlying indicator for drug resistance and survival of breast cancer,which might guide the choice of therapeutic strategies.
基金supported by the National Natural Science Foundation of China(No. 81071422)
文摘Objective： Lysosome associated protein transmembrane 4 beta （LAPTM4B） was originally identified as a gene in human hepatocellular carcinoma （HCC）. It was successfully cloned by fluorescence differential display, rapid amplification of cDNA ends （RACE） and reverse transcription polymerase chain reaction （RT-PCR）. Previous study showed that the novel gene played an important role in the occurrence, development, migration and prognosis of tumors. Pancreatic cancer is an aggressive malignancy with the majority of patients dying within one year after diagnosis. This study tries to find out the relationship between lysosome associated protein transmembrane 4 beta gene polymorphism and the susceptibility of pancreatic cancer. Methods： A case-control study was conducted in China, including 58 pancreatic cancer cases and 156 healthy controls. Human genomic DNA was used as the template, polymerase chain reaction （PCR） was used to detect the distribution of LAPTM4B genotype. Analyses Odds ratio （OR） and corresponding 95% confidence interval （95%CI） with logistic regression were performed. Results： Two alleles of LAPTM4B generated three kinds of genotypes in population, ＊1/1, ＊1/2, and ＊2/2. The genotype frequency of ＊1/1, ＊1/2 and ＊2/2 in the pancreatic cancer group were 41.4%, 44.8% and 13.8% respectively, which were not significantly different from those of healthy group （47.4%, 42.9%, 9.6%） （P=0.773, P=0.291）. Also the ＊2 allele frequency of LAPTM4B among pancreatic cancer had no significantly difference with the controls （P=0.354）. When compared to the ＊1 allele, the people with ＊2 allele had no increased risk of pancreatic cancer. Conclusion： The gene polymorphism of LAPTM4B may not influence the susceptibility of pancreatic cancer.
基金The project supported by the National Research Foundation of Korea(NRF)grant funded by the Korean government(MSIP)〔2011-0030043(SRC)〕a grant of the Korean Health Technology R&D Progect,Ministry of Health&Welfare(HI14C2230)
文摘OBJECTIVE Chloroquine is considered as a potential chemotherapy and radiotherapy sensitizer,but the anticancer effect of chloroquine alone is limited.Since we found that the flavonoid kaempferol effectively sensitizes glioma cells to chloroquine-mediated cell death,we investigated the underlying mechanisms of glioma cell death induced by the combination of kaempferol and chloroquine.METHODS To examine the effect of kaempferol and/or chloroquine on various glioma cells,cell viability assay using calcein-AM and EthD-1was performed.The changes in the lysosomal structures following treatment with kaempferol and/or chloroquine were observed by electron microscopy and fluorescence microscopy using acridine orange or Lyso-tracker Red.The changes in cathepsin D proteins were analyzed by Western blotting,immunocytochemistry,and fluorescence microscopy using BODIPY FL-pepstatin.RESULTS Treatment with subtoxic doses of chloroquine,when combined with kaempferol,effectively induced cell death in various glioma cells,but not in normal astrocytes.While kaempferol treatment increased the numbers of lysosome,chloroquine treatment increased lysosomal masses.Combined treatment with kaempferol and chloroquine induced the expansion and subsequent rupture of lysosomes,leading to the spillage of the lysosomal contents into the cytosol.We found that while kaemfperol treatment increased the active mature forms of cathepsin D,chloroquine treatment completely blocked the processing of cathepsin D.The processing of cathepsin D was also blocked by the combined treatment,but the activity of cathepsin D,which was released from the lysosomes,was restored.The cell death induced by kaempferol and chloroquine in U251 MG cells was accompanied by mitochondrial dysfunction,ER stress,and DNA damage.CONCLUSION Disruption of lysosomal membrane integrity and a resultant release of lysosomal proteases may critically contribute to the irreparable damage of various organelles and glioma cell death by chloroquine plus kaempferol.
文摘OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.
基金This project was supported by a grant from the National Natural Science Foundation of China (No. C30100169).
文摘Summary: The expression of synaptotagmin Ⅱ(Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL. The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank : NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBL-Syt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.
文摘Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)—susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core—carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.
文摘The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosisinducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I （PCD I, apoptosis） and PCD II （autophagy）-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atgl2-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class Ⅲ inhibitor, 3-methyladenine （3-MA）, and by the vacuole H＋-ATPase inhibitor, bafilomycin-A1 （Baf-A1）. S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, ATM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species （ROS） generation, and partially pro- tected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated ceils. In addition, either ATM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.
基金supported by the National Natural Science Foundation of China(No.21673037)。
文摘Carbon-based fluorescent nanomaterials have gained much attention in recent years.In this work,greenphotoluminescent carbon nanodots(CNDs;also termed carbon dots,CDs)with amine termination were synthesized via the hydrothermal treatment of amine-containing spermine and rose bengal(RB)molecules.The CNDs have an ultrasmall size of~2.2 nm and present bright photoluminescence with a high quantum yield of~80%which is possibly attributed to the loss of halogen atoms(Cl and I)during the hydrothermal reaction.Different from most CNDs which have multicolor fluorescence emission,the asprepared CNDs possess excitation-independent emission property,which can avoid fluorescence overlap with other fluorescent dyes.Moreover,the weakly basic amine-terminated surface endows the CNDs with the acidotropic effect.As a result,the CNDs can accumulate in the acidic lysosomes after cellular internalization and can serve as a favorable agent for lysosome imaging.Besides,the CNDs have a negligible impact on the lysosomal morphology even after 48 h incubation and exhibit excellent biocompatibility in the used cell models.
基金supported by the National Natural Science Foundation of China Fund(No 81541060)Science and Technology Projects from the Science Technology and Innovation Committee of Shenzhen Municipality(grant no.JCJY20170818110340383 and JCJY20170307163529489)。
文摘Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the study,a novel HIF-1αASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA.The physicochemical properties of nanocomposite were characterized using TEM,FTIR,DLS and zeta potentials.The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm,and according to the agarose gel retardation assay,the loading amount of ASODN accounted for 166.7 mg/g.The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells.The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL.The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite.In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit m RNA expression and HIF-1αprotein translation at different levels.In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue.In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%.These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.
基金Natural Science Foundation of China(NSFC,No.82001981)the fifth phase of"333 High-level Talent Cultivation Project"in Jiangsu Province(No.1092000102)the Fundamental Research Funds for the Central Universities(No.2632022ZD01)。
文摘Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organellespecific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe(BLT) with lysosomes localization capability was developed.The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable p Kavalue has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced(APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.
文摘Targeted protein degradation(TPD)has transformed drug discovery by eliminating disease-causing proteins rather than merely inhibiting their activity.Proteolysis-targeting chimeras(PROTACs)have significantly advanced this field by using bifunctional small molecules to recruit E3 ubiquitin ligases to degrade proteins of interest.However,PROTACs,relying on the ubiquitin-proteasome system,predominantly target cytosolic and nuclear proteins,but they struggle to degrade membrane or extracellular proteins.To address this gap,scientists have developed lysosome-targeting chimeras(LYTACs),which consist of an antibody or peptide binding the target protein,linked to a ligand that binds a cellsurface lysosomal trafficking receptor.By bridging a target protein on the cell surface to a lysosome-shuttling receptor,LYTACs are internalized into the cell and delivered to lysosomes,where acidic enzymes degrade various membrane proteins.In essence,LYTACs broaden the druggable proteome,allowing researchers to modulate“undruggable”targets that PROTACs and traditional inhibitors cannot touch.
