Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets.

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis. In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent. In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the Golgi apparatus independently of their clathrin adaptor binding motif.

To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

A pan-cancer analysis of prognostic significance and immunological role of lysosomal-associated membrane protein 3.

Lysosomal dysfunction can drive carcinogenesis. Lysosomal-associated membrane protein 3 (LAMP3), is a member of the Lysosome Associated Membrane Proteins and is involved in the malignant phenotype such as tumour metastasis and drug resistance, while the mechanisms that regulate the malignant progression of tumour remain vague. Our study aims to provide a more systematic and comprehensive understanding of the role of LAMP3 in the progression of various cancers by various databases.We explored the role of LAMP3 in pan-cancer using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) database. Multiple online web platforms and software were used for data analysis, including HPA, TIMER, TISIDB, GEPIA, UALCAN, Kaplan-Meier plotter, DAVID and TIGER. The immunohistochemistry was used to quantify the LAMP3 and PD-L1 expression levels in cancer.High LAMP3 expression was found in most cancers and differentially expressed across molecular and immune subtypes. The expression of LAMP3 was involved in the immune-associated processes of Antigen processing and presentation, Th17 cell differentiation, Th1 and Th2 cell differentiation, and the immune-associated pathways of T cell receptor and B cell receptor signalling pathways in most cancers. It also correlated with genetic markers of immunomodulators in various cancers. LAMP3 and PD-L1 expression in BRCA and HNSC tissues was higher than that in corresponding adjacent normal tissues by immunohistochemistry. There is a significant correlation between the expression of LAMP3 and PD-L1.Our study elucidates that LAMP3 has different expression patterns and genetic alteration patterns in different tumours. It is a potential biomarker for immune-related cancer diagnosis, prognosis and efficacy prediction.

Characterization of two human induced pluripotent stem cell lines derived from Batten disease patient fibroblasts harbouring CLN5 mutations.

The neuronal ceroid lipofuscinoses (NCLs) are a group of common inherited neurodegenerative disorders of childhood. All forms of NCLs are life-limiting with no curative treatments. Most of the 13 NCL genes encode proteins residing in endolysosomal pathways, such as CLN5, a potential lysosomal enzyme. Two induced pluripotent stem cell lines (hiPSCs) were generated from skin fibroblasts of CLN5 disease patients via non-integrating Sendai virus reprogramming. They demonstrate typical stem cell morphology, express pluripotency markers, exhibit trilineage differentiation potential and also successfully differentiate into neurons. These hiPSCs represent a potential resource to model CLN5 disease in a human context and investigate potential therapies.

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.

LIMP-2 enhances cancer stem-like cell properties by promoting autophagy-induced GSK3β degradation in head and neck squamous cell carcinoma / 国际口腔科学杂志·英文版

Cancer stem cell-like cells (CSCs) play an integral role in the heterogeneity, metastasis, and treatment resistance of head and neck squamous cell carcinoma (HNSCC) due to their high tumor initiation capacity and plasticity. Here, we identified a candidate gene named LIMP-2 as a novel therapeutic target regulating HNSCC progression and CSC properties. The high expression of LIMP-2 in HNSCC patients suggested a poor prognosis and potential immunotherapy resistance. Functionally, LIMP-2 can facilitate autolysosome formation to promote autophagic flux. LIMP-2 knockdown inhibits autophagic flux and reduces the tumorigenic ability of HNSCC. Further mechanistic studies suggest that enhanced autophagy helps HNSCC maintain stemness and promotes degradation of GSK3β, which in turn facilitates nuclear translocation of β-catenin and transcription of downstream target genes. In conclusion, this study reveals LIMP-2 as a novel prospective therapeutic target for HNSCC and provides evidence for a link between autophagy, CSC, and immunotherapy resistance.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

Targeted binding of estradiol with ESR1 promotes proliferation of human chondrocytes by inhibiting activation of ERK signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism.@*METHODS@#The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR.@*RESULTS@#Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis.@*CONCLUSIONS@#The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.

Expression of Lysosomal Membrane Proteins LAMP1, TPC1 and TPC2 in Acute Myeloid Leukemia Cells and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To investigate the relationship between the expression of lysosomal membrane proteins LAMP1, TPC1 and TPC2 in acute myeloid leukemia (AML) cells and clinical indications of AML and to explore the possible role in the genesis and development of AML and clinical significance.@*METHODS@#Real-time quantitative PCR was used to detect the mRNA expression of LAMP1, TPC1 and TPC2 in AML cell lines (HL-60, NB4) and 57 patients with acute myeloid leukemia (including 44 initially treated patients and 13 relapsed and refractory patients). The relationship of mRNA expression levels with clinical indicators and post-chemotherapy remission was analyzed.@*RESULTS@#Compared with CD34 hematopoietic stem cells (HSC), the expression levels of LAMP1 and TPC1 in AML cell lines HL-60 and NB4 significantly increased, while the expression level of TPC2 was not significantly different. The expression levels of LAMP1, TPC1 and TPC2 in bone marrow mononuclear cells (BMMNC) of AML patients were higher than those in normal human BMMNC (P90%) were also high. There was no significant difference in the expression of LAMP1, TPC1 and TPC2 between CD34HSC of patients with AML and relapsed/refractory patients (P>0.05). No correlation was found between age, sex and genotype and expression of membrane proteins (P>0.05). The expression levels of LAMP1 and TPC1 positively correlated with the number of white blood cells in peripheral blood of patients (P<0.01). LAMP1 and TPC2 were found to be associated with remission after a course of chemotherapy in newly diagnosed patients. Initially treated patients with high expression of LAMP1 in the bone marrow not easily relieved after one course of chemotherapy. Patients with high expression of TPC2 in the bone marrow more likely to be relieved after one course of chemotherapy.@*CONCLUSION@#The mRNA of the three membrane proteins are highly expressed in AML patients, and LAMP1 and TPC1 are risk factors for AML disease progression. High expression of TPC2 is beneficial for chemotherapy of patients with newly diagnosed AML.

Relationships between Rapid Eye Movement Sleep Behavior Disorder and Neurodegenerative Diseases: Clinical Assessments, Biomarkers, and Treatment / 中华医学杂志(英文版)

<p><b>Objective</b>Rapid eye movement sleep behavior disorder (RBD) is characterized by dream enactment and loss of muscle atonia during rapid eye movement sleep. RBD is closely related to α-synucleinopathies including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Many studies have investigated the markers of imaging and neurophysiological, genetic, cognitive, autonomic function of RBD and their predictive value for neurodegenerative diseases. This report reviewed the progress of these studies and discussed their limitations and future research directions.</p><p><b>Data Sources</b>Using the combined keywords: "RBD", "neurodegenerative disease", "Parkinson disease", and "magnetic resonance imaging", the PubMed/MEDLINE literature search was conducted up to January 1, 2018.</p><p><b>Study Selection</b>A total of 150 published articles were initially identified citations. Of the 150 articles, 92 articles were selected after further detailed review. This study referred to all the important English literature in full.</p><p><b>Results</b>Single-nucleotide polymorphisms in SCARB2 (rs6812193) and MAPT (rs12185268) were significantly associated with RBD. The olfactory loss, autonomic dysfunction, marked electroencephalogram slowing during both wakefulness and rapid eye movement sleep, and cognitive impairments were potential predictive markers for RBD conversion to neurodegenerative diseases. Traditional structural imaging studies reported relatively inconsistent results, whereas reduced functional connectivity between the left putamen and substantia nigra and dopamine transporter uptake demonstrated by functional imaging techniques were relatively consistent findings.</p><p><b>Conclusions</b>More longitudinal studies should be conducted to evaluate the predictive value of biomarkers of RBD. Moreover, because the glucose and dopamine metabolisms are not specific for assessing cognitive cognition, the molecular metabolism directly related to cognition should be investigated. There is a need for more treatment trials to determine the effectiveness of interventions of RBD on preventing the conversion to neurodegenerative diseases.</p>

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis

BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.

Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

OBJECTIVES: There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. METHODS: It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. RESULTS: The result showed 14 up-regulated (1.5-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. CONCLUSIONS: It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome.

Expression of EV71-VP1, PSGL-1 and SCARB2 in Tissues of Infants with Brain Stem Encephalitis / 法医学杂志

OBJECTIVE@#To understand the correlation of enterovirus 71 (EV71), P-selectin glycoprotein ligand-1 (PSGL-1), and scavenger receptor B2 (SCARB2) and to explore the possible pathway and mechanism of EV71 infection by observing the expression of EV71, PSGL-1 and SCARB2 in tissues of infants with brain stem encephalitis.@*METHODS@#The organs and tissues of infants with EV71-VP1 positivity in their brain stems were chosen. Expression and distribution of EV71-VP1, PSGL-1, and SCARB2 were detected and compared by immunohistochemistry.@*RESULTS@#Strong staining of EV71 -VP1 was observed in the neuron, glial cells, the inflammatory cells of perivascular cuffing, parietal cells of the gastric fundus gland while alveolar macrophages, intestinal gland epithelium cells, mucosa lymphoid nodule and lymphocyte of palatine tonsil showed moderate staining and weak staining were displayed in mesenteric lymph nodes and lymphocyte of spleen. PSGL-1 expression was detected in parietal cells of the gastric fundus gland, tonsillar crypt squamous epithelium, alveolar macrophages and leukocytes in each tissue. SCARB2 expression was observed in all the above tissues except the intestines and spleen.@*CONCLUSION@#The distribution of EV71 correlates with SCARB2 expression. SCARB2 plays an important role in virus infection and replication. Stomach may be an important site for EV71 replication.

Advances in research of SCARB2 functions and related disorders / 中华医学遗传学杂志

SCARB2 (scavenger receptor class B, member 2) is a lysosomal membrane glucoprotein, which is encoded by SCARB2 gene. It takes vital parts in the physiological and pathological processes including the transportation of beta-glucocerebrosidase to the lysosome, infection of EV71 and load-induced cardiac myocyte hypertrophy. This article has reviewed the molecular structure and functions of SCARB2 gene and its protein, as well as their relationship with diseases.

Immunohistochemical study of amelogenin and lysosome-associate membrane proteins (LAMPs) in cartilage / Estudio inmunohistoquímico de la amelogenina y proteínas de membrana asociadas a lisosomas (LAMPs) en el cartílago

Amelogenin is one of the enamel matrix proteins secreted by ameloblasts during enamel formation in tooth development. Recent studies showed that the amelogenin is expressed in chondrocyte. Lysosome-associated membrane proteins (LAMPs) have been identified as binding partner proteins to amelogenin and it has been suggested they act as signaling receptors of amelogenin. The purpose of this study is to clarify the localization of amelogenin and LAMPs in growth plate cartilage and cartilaginous nodules in micromass culture. Mouse knee joints including tibia growth plate at 4 weeks old and micromass cultures of limb bud mesenchymal cells after 2 weeks were fixed in paraformaldehyde, routinely processed, sections were cut and immunostained with amelogenin, collagen type II and type X, LAMP-1 and -3. The positive immunoreaction of amelogenin was observed both in proliferation and hypertrophic zone cartilage of growth plate after enzymatic pretreatment in immunostaining. Furthermore, cartilaginous nodules in micromass culture were immunopositive to amelogenin. The chondrocytes in the proliferation zone of the growth plate were immunopositive to LAMP-1 but weakly stained in the chondrocytes of hypertrophic zone. These observations indicate that amelogenin may be present in cartilage matrix produced in vivo and in vitro and amelogenin may involve cartilage formation through the LAMP-1 signaling pathway.

La amelogenina es una de las proteínas de la matriz del esmalte secretadas por ameloblastos durante la formación del esmalte en el desarrollo dentario. Estudios recientes demuestran que la amelogenina se expresa en los condrocitos. Las proteínas de membrana asociadas a lisosomas (LAMPs) se han identificado como proteínas de unión asociadas a la amelogenina; se ha sugerido que actúan como receptores de señalización de la amelogenina. El propósito de este estudio fue aclarar la localización de la amelogenina y las LAMPs en el cartílago de crecimiento y nódulos cartilaginosos en cultivos de micromasa. Articulaciones de la rodilla del ratón, que incluían la placa de crecimiento tibial de 4 semanas de edad y cultivos de micromasa de células mesenquimales del brote del miembro después de 2 semanas se fijaron en paraformaldehído y procesaron rutinariamente. Los cortes fueron sometidos a inmunotinción con amelogenina, colágeno tipo II y X, LAMP-1 y LAMP-3 . Se observó inmunorreacción positiva de amelogenina tanto en la zona proliferación e hipertrófica del cartílago de crecimiento después del pretratamiento enzimático. Además, los nódulos cartilaginosos en el cultivo de micromasa eran inmunopositivos para la amelogenina. Los condrocitos en la zona de proliferación de la placa de crecimiento fueron immunopositivos a LAMP-1, mientras que los condrocitos de la zona hipertrófica se tiñeron débilmente. Estas observaciones indican que la amelogenina puede estar presente en la matriz del cartílago producida tanto in vivo e in vitro, además la amelogenina puede estar implicada en la formación de cartílago mediante la vía de señalización de LAMP-1.

Biomarcadores proteómicos en la enfermedad de Parkinson / Proteomic biomarkers in Parkinson's disease

La enfermedad de Parkinson (EP) es un trastorno neurodegenerativo que afecta al movimiento, cuya causa es la muerte de las neuronas dopaminérgicas de la parte compacta de la sustancia negra. El diagnóstico es fundamentalmente clínico, pero, a pesar de que los signos y síntomas de la EP se conocen bien, el error diagnóstico es relativamente alto. Se calcula que un 10-30% de los pacientes inicialmente diagnosticados de EP se reclasifican posteriormente. Esta enfermedad tiene una alta prevalencia a partir de los 60 años, y uno de los mayores problemas que tiene es que su diagnóstico se hace cuando el proceso degenerativo está muy avanzado. Por tanto, es necesaria la búsqueda de biomarcadores que permitan un diagnóstico precoz de la EP, seguir su progresión, diferenciarla de otras patologías relacionadas (parkinsonismos) y que ayuden a monitorizar el efecto de nuevas terapias. El hecho de que existan mutaciones que conducen a la EP, así como combinaciones poligénicas que pueden actuar como factores de riesgo, sugiere que es posible analizar las proteínas resultantes de la expresión de estos genes en tejidos periféricos, que, una vez demostrada su sensibilidad y especificidad, podrían utilizarse como biomarcadores de la EP, incluso en fases iniciales de la enfermedad. El objetivo del presente trabajo es centrarse en una revisión detallada de los principales biomarcadores proteómicos candidatos investigados hasta el momento, discutiendo la literatura más reciente (AU)

Parkinson’s disease (PD) is a neurodegenerative disorder that affects movement and is caused by the death of the dopaminergic neurons in the compact part of the substantia nigra. Its diagnosis is essentially clinical, but although the signs and symptoms of PD are well known, the rate of diagnostic error is relatively high. It is estimated that 10-30% of patients initially diagnosed with PD are later reclassified. This disease has a high prevalence beyond the age of 60, and one of its biggest problems is that it is diagnosed when the degenerative process is already at a very advanced stage. Therefore, it is necessary to look for other biomarkers that make it possible to carry out an early diagnosis of PD, follow up its development, distinguish it from other related pathologies (parkinsonisms) and help monitor the effect of novel therapies. The fact that there are mutations that lead to PD, as well as polygenetic combinations that can act as risk factors, suggests the possibility of measuring the proteins resulting from the expression of these genes in peripheral tissues. And once their sensitivity and specificity have been proved they could be used as biomarkers for PD, even in the early phases of the disease. The aim of this work is to focus on a detailed review of the main candidate proteomic biomarkers researched to date by discussing the most recent literature (AU)