Lysosomal membrane protein Sidt2 knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway / 南方医科大学学报

OBJECTIVE@#To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout.@*METHODS@#The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed.@*RESULTS@#Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells.@*CONCLUSION@#Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.

Effect of Porphyromonas gingivalis infection on IFNGR1 palmitoylation in esophageal cancer cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Porphyromonas gingivalis (Pg) infection on IFNGR1 palmitoylation and biological behaviors of esophageal squamous cell carcinoma (ESCC) cells and the clinical implications.@*METHODS@#The expression levels of IFNGR1 protein in ESCC cell lines KYSE30 and KYSE70 were detected using Western blotting at 24 and 48 h after Pg infection, and 2-BP was used to detect IFNGR1 palmitoylation in the cells. KYSE70 cells with wild-type IFNGR1 (IFNGR1-WT cells) and with IFNGR1-C122A palmitoylation site mutation induced by site-specific mutagenesis (IFNGR1-C122A cells) were both infected with Pg, and the changes in palmitoylation of IFNGR1-C122A were analyzed using immunofluorescence and Click-iT assays. The changes in proliferation, migration and invasion ability of the infected cells were evaluated using plate cloning assay, scratch assay and Transwell assay, and IFNGR1 co-localization with lysosomal marker LAMP2 was dected using immunofluorescence assay. Immunohistochemistry was used to detect Pg infection and IFNGR1 protein expression in 50 ESCC tissues, and their correlation with the clinicopathological characteristics and survival outcomes of the patients was analyzed.@*RESULTS@#Pg infection down-regulated the protein expression of IFNGR1 in ESCC and promoted IFNGR1 palmitoylation at site 122. In IFNGR1-WT cells, Pg infection significantly enhanced cell proliferation, migration and invasion (P < 0.05). Similarly, Pg also significantly promoted proliferation, migration and invasion of IFNGR1-C122A cells, but to a lesser extent as compared with the wild-type cells (P < 0.05). Immunofluorescence assay showed that Pg and ZDHHC3 promoted IFNGR1 degradation within the lysosome. Immunohistochemical studies of the ESCC tissue samples showed a negative correlation between IFNGR1 and Pg expression, and a reduced IFNGR1 expression was correlated with a poorer survival outcome of the patient.@*CONCLUSION@#Pg infection enhances IFNGR1 palmitoylation to promote progression of ESCC, and elimination of Pg and inhibiting IFNGR1 palmitoylation may effectively control ESCC progression.

Analysis of lysosomal enzyme activity and genetic variants in a child with late-onset Pompe disease / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical features, lysosomal enzymatic [acid α-glucosidase (GAA)] activities and genetic variants in a child with late-onset Pompe disease (LOPD).@*METHODS@#Clinical data of a child who had presented at the Genetic Counseling Clinic of West China Second University Hospital in August 2020 was retrospectively analyzed. Blood samples were collected from the patient and her parents for the isolation of leukocytes and lymphocytes as well as DNA extraction. The activity of lysosomal enzyme GAA in leukocytes and lymphocytes was analyzed with or without addition of inhibitor of GAA isozyme. Potential variants in genes associated with neuromuscular disorders were analyzed, in addition with conservation of the variant sites and protein structure. The remaining samples from 20 individuals undergoing peripheral blood lymphocyte chromosomal karyotyping were mixed and used as the normal reference for the enzymatic activities.@*RESULTS@#The child, a 9-year-old female, had featured delayed language and motor development from 2 years and 11 months. Physical examination revealed unstable walking, difficulty in going upstairs and obvious scoliosis. Her serum creatine kinase was significantly increased, along with abnormal electromyography, whilst no abnormality was found by cardiac ultrasound. Genetic testing revealed that she has harbored compound heterozygous variants of the GAA gene, namely c.1996dupG (p.A666Gfs*71) (maternal) and c.701C>T (p.T234M) (paternal). Based on the guidelines from the American College of Medical Genetics and Genomics, the c.1996dupG (p.A666Gfs*71) was rated as pathogenic (PVS1+PM2_Supporting+PM3), whilst the c.701C>T (p.T234M) was rated as likely pathogenic (PM1+PM2_Supporting+PM3+PM5+PP3). The GAA in the leukocytes from the patient, her father and mother were respectively 76.1%, 91.3% and 95.6% of the normal value without the inhibitor, and 70.8%, 112.9% and 128.2% of the normal value with the inhibitor, whilst the activity of GAA in their leukocytes had decreased by 6 ~ 9 times after adding the inhibitor. GAA in lymphocytes of the patient, her father and mother were 68.3%, 59.0% and 59.5% of the normal value without the inhibitor, and 41.0%, 89.5% and 57.7% of the normal value with the inhibitor, the activity of GAA in lymphocytes has decreased by 2 ~ 5 times after adding the inhibitor.@*CONCLUSION@#The child was diagnosed with LOPD due to the c.1996dupG and c.701C>T compound heterozygous variants of the GAA gene. The residual activity of GAA among LOPD patients can range widely and the changes may be atypical. The diagnosis of LOPD should not be based solely on the results of enzymatic activity but combined clinical manifestation, genetic testing and measurement of enzymatic activity.

A dynamically evolving war between autophagy and pathogenic microorganisms / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Autophagy is an intracellular degradation process that maintains cellular homeostasis. It is essential for protecting organisms from environmental stress. Autophagy can help the host to eliminate invading pathogens, including bacteria, viruses, fungi, and parasites. However, pathogens have evolved multiple strategies to interfere with autophagic signaling pathways or inhibit the fusion of autophagosomes with lysosomes to form autolysosomes. Moreover, host cell matrix degradation by different types of autophagy can be used for the proliferation and reproduction of pathogens. Thus, determining the roles and mechanisms of autophagy during pathogen infections will promote understanding of the mechanisms of pathogen‍‒‍host interactions and provide new strategies for the treatment of infectious diseases.

The new target of Rapamycin: lysosomal calcium channel TRPML1 / 生理学报

Rapamycin (Rap) is an immunosuppressant, which is mainly used in the anti-rejection of organ transplantation. Meanwhile, it also shows great potential in the fields of anticancer, neuroprotection and anti-aging. Rap can inhibit the activity of mammalian target of Rap (mTOR). It activates the transcription factor EB (TFEB) to up-regulate lysosomal function and eliminates the inhibitory effect of mTOR on ULK1 (unc-51 like autophagy activating kinase 1) to promote autophagy. Recent research showed that Rap can directly activate the lysosomal cation channel TRPML1 in an mTOR-independent manner. TRPML1 activation releases lysosomal calcium. Calcineurin functions as the sensor of the lysosomal calcium signal and activates TFEB, thus promoting lysosome function and autophagy. This finding has greatly broadened and deepened our understanding of the pharmacological roles of Rap. In this review, we briefly introduce the canonical Rap-mTOR-ULK1/TFEB signaling pathway, and then discuss the discovery of TRPML1 as a new target of Rap and the pharmacological potential of this novel Rap-TRPML1-Calcineurin-TFEB pathway.

Regulation of FN1 degradation by the p62/SQSTM1-dependent autophagy-lysosome pathway in HNSCC / 国际口腔科学杂志·英文版

Epithelial-mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle's balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy-lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy-lysosome degradation mechanism of FN1.

Preparation of curcumin TPP-PEG-PE nanomicelles with mitochondrial targeting and lysosomal escape functions and its effect on promoting breast cancer cell apoptosis / 中国中药杂志

Orthogonal experiments were used to optimize the process parameters of curcumin TPP-PEG-PCL nanomicelles; the particle size, electric potential and morphology under the electron microscope were systematically detected for the curcumin TPP-PEG-PCL nanomicelles; and the stability and in vitro release of the curcumin TPP-PEG-PCL nanomicelles were investigated. With DID fluorescent dye as the fluorescent probe, flow cytometry was used to study the uptake of nanomicelles by breast cancer cells, and laser confocal microscopy was used to study the mitochondrial targeting and lysosomal escape functions of nanomicelles. Under the same dosage conditions, the effect of curcumin TPP-PEG-PCL nanomicelles on promoting the apoptosis of breast cancer cells was evaluated. The optimal particle size of curcumin TPP-PEG-PCL nanomicelle was(17.3±0.3) nm, and the Zeta potential was(14.6±2.6) mV in orthogonal test. Under such conditions, the micelle appeared as regular spheres under the transmission electron microscope. Fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote drug uptake by tumor cells, escape from lysosomal phagocytosis, and target the mitochondria. The cell survival rate and Hoechst staining positive test results showed that curcumin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of breast cancer cells. The curcumin TPP-PEG-PCL micelles can significantly reduce the mitochondrial membrane potential of breast cancer cells, increase the release of cytochrome C, significantly increase the expression of pro-apoptotic protein Bcl-2 and reduce the expression of anti-apoptotic Bax protein. These test results were significantly better than those of curcumin PEG-PCL nanomicelles and curcumin, with statistically significant differences. The results revealed that curcumin TPP-PEG-PCL nanomicelles can well target breast cancer cell mitochondria and escape from the lysosomal capture, thereby enhancing the drug's role in promoting tumor cell apoptosis.

Role of endocytosis in cell surface CXC chemokine receptor 4 expression of stem cells from apical papilla / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the change of cell surface CXC chemokine receptor 4 (CXCR4) expression of stem cells from apical papilla (SCAP) after the inhibition of endocytotic pathway, thus to provide experimental basis for the mechanism of SCAP migration.@*METHODS@#The immunofluorescence analysis was conducted to examine the co-expression of CXCR4 and endocytotic compartments, including early endosomes, recycling endosomes and lysosomes in SCAP. Several Rab proteins were applied as markers of organelles in the endocytotic pathway, including Rab5 for early endosomes, Rab11A for recycling endosomes, and Lamp1 for lysosomes. The co-localization of CXCR4 with these endodontic compartments was further observed by proximity ligation assay (PLA). SCAP was treated with two kinds of endocytotic inhibitors, Blebbistatin and Dynasore, at a concentration of 80 μmol/L, respectively. The conditioning time was 1 hour. Flow cytometry was carried out to evaluate the proportion of SCAP that expressed CXCR4 on cell surface. The data were analysed by analysis of variance (ANOVA).@*RESULTS@#The red staining of CXCR4 on immunofluorescence confocal microscopy predominantly overlapped with the green staining of Rab5 and Rab11A, and partly overlapped with Lamp1. It indicated that most CXCR4 molecules were located in early endosomes and recycling endosomes, and some were located in lysosomes. The PLA results revealed that the co-localizaiton of CXCR4 with endocytotic compartments could be observed in early endosomes, recycling endosomes and lysosomes. According to the results of flow cytometry, the proportion of SCAP that expressed CXCR4 on cell surface was as low as 0.13%±0.10%. After the inhibition of endocytosis by pretreating the cells with the following two inhibitors, Blebbistatin and Dynasore, the percentage of SCAP that positively expressed CXCR4 on cell surface was significantly increased to 13.34%±1.31% in Blebbistatin group and 4.03%±0.92% in Dynasore group (F=16.721, P<0.001). Moreover, the number of SCAP that expressed CXCR4 on cell surface in Blebbistatin group was significantly higher than that in Dynasore group (P<0.001).@*CONCLUSION@#The inhibition of endocytotic pathway could increase the number of SCAP that expressed CXCR4 on cell surface, and provide potency for the migration of SCAP.

Birth of a healthy baby after preimplantation genetic diagnosis in a carrier of mucopolysaccharidosis type II: The first case in Korea / 대한생식의학회지

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive lysosomal storage disease caused by mutation of the iduronate-2-sulfatase gene. The mutation results in iduronate-2-sulfatase deficiency, which causes the progressive accumulation of heparan sulfate and dermatan sulfate in cellular lysosomes. The phenotype, age of onset, and symptoms of MPS II vary; accordingly, the disease can be classified into either the early-onset type or the late-onset type, depending on the age of onset and the severity of the symptoms. In patients with severe MPS II, symptoms typically first appear between 2 and 5 years of age. Patients with severe MPS II usually die in the second decade of life although some patients with less severe disease have survived into their fifth or sixth decade. Here, we report the establishment of a preimplantation genetic diagnosis (PGD) strategy using multiplex nested polymerase chain reaction, direct sequencing, and linkage analysis. Unaffected embryos were selected via the diagnosis of a single blastomere, and a healthy boy was delivered by a female carrier of MPS II. This is the first successful application of PGD in a patient with MPS II in Korea

Neuroprotective Autophagic Flux Induced by Hyperbaric Oxygen Preconditioning is Mediated by Cystatin C / 神经科学通报·英文版

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.