Charged Multivesicular Body Protein Research Articles

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

ABSTRACTTo provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

The ESCRT-III molecules regulate the apical targeting of bile salt export pump

BackgroundThe bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting.MethodsThe human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP.ResultsWe identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP.ConclusionsOur results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.

Metformin-Enhanced Cardiac AMP-Activated Protein Kinase/Atrogin-1 Pathways Inhibit Charged Multivesicular Body Protein 2B Accumulation in Ischemia-Reperfusion Injury.

Background: Cardiac autophagic flux is impaired during myocardial ischemia/reperfusion (MI/R). Impaired autophagic flux may exacerbate MI/R injury. Charged multivesicular body protein 2B (CHMP2B) is a subunit of the endosomal sorting complex required for transport (ESCRT-III) complex that is required for autophagy. However, the reverse role of CHMP2B accumulation in autophagy and MI/R injury has not been established. The objective of this article is to elucidate the roles of AMP-activated protein kinase (AMPK)/atrogin-1 pathways in inhibiting CHMP2B accumulation in ischemia–reperfusion injury.Methods: Male C57BL/6 mice (3–4 months) and H9c2 cardiomyocytes were used to evaluate MI/R and hypoxia/reoxygenation (H/R) injury in vivo and in vitro, respectively. MI/R was built by a left lateral thoracotomy and occluded the left anterior descending artery. H9c2 cells were firstly treated in 95% N2 and 5% CO2 for 15 h and reoxygenation for 1 h. Metformin (100 mg/kg/d) and CHMP2B (Ad-CHMP2B) transfected adenoviruses were administered to the mice. The H9c2 cells were treated with metformin (2.5 mM), MG-132 (10 μM), bafilomycin A1 (10 nM), and compound C (20 μM).Results: Autophagic flux was found to be inhibited in H/R-treated cardiomyocytes and MI/R mice, with elevated cardiac CHMP2B accumulation. Upregulated CHMP2B levels in the in vivo and in vitro experiments were shown to inhibit autophagic flux leading to the deterioration of H/R-cardiomyocytes and MI/R injury. This finding implies that CHMP2B accumulation increases the risk of myocardial ischemia. Metformin suppressed CHMP2B accumulation and ameliorated H/R-induced autophagic dysfunction by activating AMPK. Activated AMPK upregulated the messenger RNA expression and protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in the myocardium. Atrogin-1 significantly enhanced the interaction between atrogin-1 and CHMP2B, therefore, promoting CHMP2B degradation in the MI/R myocardium. Finally, this study revealed that metformin-inhibited CHMP2B accumulation induced autophagic impairment and ischemic susceptibility in vivo through the AMPK-regulated CHMP2B degradation by atrogin-1.Conclusion: Impaired CHMP2B clearance in vitro and in vivo inhibits autophagic flux and weakens the myocardial ischemic tolerance. Metformin treatment degrades CHMP2B through the AMPK-atrogin-1-dependent pathway to maintain the homeostasis of autophagic flux. This is a novel mechanism that enriches the understanding of cardioprotection.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (−/−) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (−/−) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (−/−) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (−/−) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (−/−) mice could serve as a mouse model for the development of therapeutic strategies.

WITHDRAWN: LINC00342 promotes thyroid carcinoma progression by targeting miR-384/CHMP5 pathway

Abstract Thyroid carcinoma (THCA) is the most common thyroid malignancy and accounts for 1% of overall malignant tumors. Long intergenic non-protein coding RNA 342 (LINC00342) has been validated to be a regulator in non-small cell lung cancer (NSCLC). However, the biological role of LINC00342 in THCA has not been reported or discussed yet. Therefore, the present study focused on the role of LINC00342 and its mechanism in THCA. Firstly, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted for detecting LINC00342 expression in THCA and the outcomes disclosed that LINC00342 had a high expression in THCA tissues and cells. Besides, functional assays reflected that LINC00342 silence inhibited THCA progression by anti-proliferation, anti-migration/invasion and pro-apoptosis as well as blocking epithelial-mesenchymal transition (EMT). Next, through RNA immunoprecipitation (RIP), RNA pull down and luciferase reporter assays, we confirmed that LINC00342 and charged multivesicular body protein 5 (CHMP5) could bind to miR-384. Further, the expression of CHMP5 was declined with miR-384 up-regulation or LINC00342 down-regulation. Finally, rescue experiments revealed that CHMP5 upregulation reversed LINC00342 knockdown-mediated effects on THCA cells. All data showed that LINC00342 promoted THCA progression by absorbing miR-384 to upregulate CHMP5 expression. These findings proved that LINC00342 might be a promising target for THCA treatment.

Differences in plasma proteomes for active tuberculosis, latent tuberculosis and non-tuberculosis mycobacterial lung disease patients with and without ESAT-6/CFP10 stimulation

BackgroundTuberculosis (TB) is one of the world’s most problematic infectious diseases. The pathogen Mycobacterium tuberculosis (Mtb) is contained by the immune system in people with latent TB infection (LTBI). No overt disease symptoms occur. The environmental and internal triggers leading to reactivation of TB are not well understood. Non-tuberculosis Mycobacteria (NTM) can also cause TB-like lung disease. Comparative analysis of blood plasma proteomes from subjects afflicted by these pathologies in an endemic setting may yield new differentiating biomarkers and insights into inflammatory and immunological responses to Mtb and NTM.MethodsBlood samples from 40 human subjects in a pastoral region of Ethiopia were treated with the ESAT-6/CFP-10 antigen cocktail to stimulate anti-Mtb and anti-NTM immune responses. In addition to those of active TB, LTBI, and NTM cohorts, samples from matched healthy control (HC) subjects were available. Following the generation of sample pools, proteomes were analyzed via LC-MS/MS. These experiments were also performed without antigen stimulation steps. Statistically significant differences using the Z-score method were determined and interpreted in the context of the proteins’ functions and their contributions to biological pathways.ResultsMore than 200 proteins were identified from unstimulated and stimulated plasma samples (UPSs and SPSs, respectively). Thirty-four and 64 proteins were differentially abundant with statistical significance (P < 0.05; Benjamini-Hochberg correction with an FDR < 0.05) comparing UPS and SPS proteomic data of four groups, respectively. Bioinformatics analysis of such proteins via the Gene Ontology Resource was indicative of changes in cellular and metabolic processes, responses to stimuli, and biological regulations. The m7GpppN-mRNA hydrolase was increased in abundance in the LTBI group compared to HC subjects. Charged multivesicular body protein 4a and platelet factor-4 were increased in abundance in NTM as compared to HC and decreased in abundance in NTM as compared to active TB. C-reactive protein, α-1-acid glycoprotein 1, sialic acid-binding Ig-like lectin 16, and vitamin K-dependent protein S were also increased (P < 0.05; fold changes≥2) in SPSs and UPSs comparing active TB with LTBI and NTM cases. These three proteins, connected in a STRING functional network, contribute to the acute phase response and influence blood coagulation.ConclusionPlasma proteomes are different comparing LTBI, TB, NTM and HC cohorts. The changes are augmented following prior blood immune cell stimulation with the ESAT-6/CFP-10 antigen cocktail. The results encourage larger-cohort studies to identify specific biomarkers to diagnose NTM infection, LTBI, and to predict the risk of TB reactivation.

Abstract LB-047: CHMP5 dependency is a therapeutic vulnerability of T cell acute lymphoblastic leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a very aggressive hematologic malignancy that results from the transformation of immature T-cell progenitors. Aberrant cell growth and proliferation is sustained by strong oncogenic drivers, notably NOTCH1 and MYC, whose activation is present in more than half of all T-ALL cases. However, although it is known that post-translational modifications, such as ubiquitination by the E3-ligase FBXW7, regulate NOTCH1 and MYC protein stability and activity, regulatory proteins involved in their stability remain unestablished. The Charged multivesicular body protein 5 (CHMP5), an accessory protein of the ESCRT (endosomal sorting complex required for transport) pathway, has been shown to regulate the stability of client proteins in thymocytes through the recruitment of de-ubiquitinating enzymes. Here, we report that CHMP5 protein is not only highly expressed in T-ALL cells, but also promotes the stability of transactivating intracellular NOTCH1 (ICN1) and MYC. We found that depletion of CHMP5 significantly reduced the abundance of ICN1 and MYC, and the transcription of their target genes in vitro, as well as suppressed the development of T-ALL in vivo in a murine model of NOTCH1 driven T-ALL. Additionally, loss of CHMP5 restored T-ALL cell susceptibility to chemotherapeutic agents, including glucocorticoids and gamma secretase inhibitors, resistance to which contributes to disease relapse in patients. We further reveal CHMP5 as an adaptor protein required to orchestrate T-ALL client protein deubiquitination and stability. Altogether, our data suggest that CHMP5 is a non-oncogenic dependency of T-ALL and its depletion may be an effective therapeutic strategy for T-ALL. Citation Format: Katharine Umphred-Wilson, Stanley Adoro. CHMP5 dependency is a therapeutic vulnerability of T cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-047.

Up-regulation of CHMP4B alleviates microglial necroptosis induced by traumatic brain injury.

Microglial cells are key component of central nervous system (CNS) and mediate the immune response of the brain under physiological or pathological conditions. It tends to activate into a pro‐inflammatory M1 phenotype after traumatic brain injury (TBI) and promote secondary brain damage. Recently, necroptosis was found to promote microglial activation and neuroinflammation after TBI. However, the mechanism and specific interventions of microglial necroptosis after TBI remain poorly investigated. Here, we reported that overexpress the charged multivesicular body protein 4b (CHMP4B) which is a core member of the endosomal sorting required for transport complex III (ESCRT‐III) significantly decreased the level of necroptosis in microglia, improved neurological function recovery and protected against cell death after TBI. Further investigation showed that forkhead transcription factor O1 (FOXO1) was a crucial transcription factor that increased CHMP4B transcription by binding to the promoter region, thereby inhibiting necroptosis in microglia. Collectively, our findings demonstrated that CHMP4B relieved microglial necroptosis and neuroinflammation after TBI, and promote the recovery of nerve function. FOXO1 is an important factor in promoting CHMP4B expression. This study provides the novel viewpoint for TBI prevention and treatment.

ESCRT Machinery Mediates Cytokinetic Abscission in the Unicellular Red Alga Cyanidioschyzon merolae.

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than in animals, the roles of ESCRT in cytokinesis are poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. merolae cells divide by furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated—a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

MiR-17 regulates the proliferation and differentiation of retinal progenitor cells by targeting CHMP1A

MicroRNAs have a vital effect on the differentiation of many types of progenitor cells. Recent studies have suggested that miR-17 plays an important role in the differentiation process of brain neural progenitor cells (NPC). Nevertheless, its detailed functions in regulating retinal progenitor cells (RPC) remain unclear. In our study, overexpression and knockdown of miR-17 were performed by transfecting RPC with mimics and inhibitors, respectively. Next, we investigated the role of miR-17 in RPC proliferation and differentiation by the following experiments: qPCR, CCK8, Edu staining, immunostaining and Western blot. The results revealed that miR-17 inhibited RPC proliferation but enhanced differentiation. Furthermore, according to a web-based database analysis, we identified charged multivesicular body protein 1A (CHMP1A) as a target gene. A dual luciferase reporter system showed that miR-17 specifically binds to the CHMP1A 3’ untranslated region (UTR). Next, our data showed upregulation of miR-17 decreased CHMP1A protein level, causing reduced proliferation and enhanced differentiation of RPC. Downregulation of miR-17 led to enhanced CHMP1A protein expression, increased RPC proliferation and decreased differentiation. Taken together, our data provide a proven pathway by which miR-17 regulates RPC proliferation and differentiation by targeting CHMP1A.

Coordinated alteration of mRNA-microRNA transcriptomes associated with exosomes and fatty acid metabolism in adipose tissue and skeletal muscle in grazing cattle.

ObjectiveOn the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach.MethodsThe subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses.ResultsThe results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The ‘Exosome’, ‘Carbohydrate metabolism’ and ‘Lipid metabolism’ were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysis-associated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms ‘Endosome’, ‘Caveola’, ‘Endocytosis’, ‘Carbohydrate metabolism’, and with pathways related to environmental information processing and the endocrine system.ConclusionExosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.

ESCRT subunit CHMP4B localizes to primary cilia and is required for the structural integrity of the ciliary membrane.

Proteins specialized in the detection, generation, or stabilization of membrane curvature play important roles in establishing various morphologies of cells and cellular organelles. Primary cilia are cellular organelles that protrude from the cell surface using a microtubule-based cytoskeleton called the axoneme as a structural support. It is unclear whether the integrity of the high curvature of the ciliary membrane depends on membrane curvature-related proteins. Charged Multivesicular Body Protein 4B (CHMP4B), a subunit of the endosomal sorting complexes required for transport (ESCRT), can stabilize membrane curvature. Here we show that CHMP4B is involved in the assembly and maintenance of primary cilia. CHMP4B was localized to primary cilia in mammalian cells. Knockdown of CHMP4B interfered with cilium assembly and also caused fragmentation of preexisting cilia. By contrast, cilium formation was unaffected by the interruption of the ESCRT-dependent endocytic degradation pathway. Morpholino (MO)-mediated CHMP4B depletion in zebrafish embryos induced characteristic phenotypes of ciliary defects such as curved body axis, hydrocephalus, otolith malformation, and kidney cyst. Our study reveals a new role for the multifunctional protein CHMP4B as a key factor in maintaining the structural integrity of primary cilia.

Structural Insights into AQP2 Targeting to Multivesicular Bodies.

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

MiR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling.

Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3'-untranslated region (3'-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3'-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.

A charged multivesicular body protein (CHMP4B) is required for lens growth and differentiation

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.

MAPT/Tau accumulation represses autophagy flux by disrupting IST1-regulated ESCRT-III complex formation: a vicious cycle in Alzheimer neurodegeneration

ABSTRACTMacroautophagy/autophagy deficit induces intracellular MAPT/tau accumulation, the hallmark pathology in Alzheimer disease (AD) and other tauopathies; however, the reverse role of MAPT accumulation in autophagy and neurodegeneration is not clear. Here, we found that overexpression of human wild-type full-length MAPT, which models MAPT pathologies as seen in sporadic AD patients, induced autophagy deficits via repression of autophagosome-lysosome fusion leading to significantly increased LC3 (microtubule-associated protein 1 light chain 3)-II and SQSTM1/p62 (sequestosome 1) protein levels with autophagosome accumulation. At the molecular level, intracellular MAPT aggregation inhibited expression of IST1 (IST1 factor associated with ESCRT-III), a positive modulator for the formation of ESCRT (the Endosomal Sorting Complex Required for Transport) complex that is required for autophagosome-lysosome fusion. Upregulating IST1 in human MAPT transgenic mice attenuated autophagy deficit with reduced MAPT aggregation and ameliorated synaptic plasticity and cognitive functions, while downregulating IST1 per se induced autophagy deficit with impaired synapse and cognitive function in naïve mice. IST1 can facilitate association of CHMP2B (charged multivesicular body protein 2B) and CHMP4B/SNF7-2 to form ESCRT-III complex, while lack of IST1 impeded the complex formation. Finally, we demonstrate that MAPT accumulation suppresses IST1 transcription with the mechanisms involving the ANP32A-regulated mask of histone acetylation. Our findings suggest that the AD-like MAPT accumulation can repress autophagosome-lysosome fusion by deregulating ANP32A-INHAT-IST1-ESCRT-III pathway, which also reveals a vicious cycle of MAPT accumulation and autophagy deficit in the chronic course of AD neurodegeneration.Abbreviations: AAV: adeno-associated virus; Aβ: β-amyloid; aCSF: artificial cerebrospinal fluid; AD: Alzheimer disease; ANP32A: acidic nuclear phosphoprotein 32 family member A; ATG: autophagy related; AVs: autophagic vacuoles; CEBPB: CCAAT enhancer binding protein beta; CHMP: charged multivesicular body protein; DMEM: Dulbecco’s modified eagle’s medium; EBSS: Earle’s balanced salt solution; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; fEPSPs: field excitatory postsynaptic potentials; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3B: glycogen synthase kinase 3 beta; HAT: histone acetyl transferase; HDAC: histone deacetylase; INHAT: inhibitor of histone acetyl transferase; IST1: IST1 factor associated with ESCRT-III; LAMP2: lysosomal associated membrane protein 2; LTP: long-term potentiation; MAP1LC3: microtubule associated protein 1 light chain 3; MAPT/tau: microtubule associated protein tau; MVB: multivesicular bodies; MWM: Morris water maze; PBS: phosphate-buffered saline solution; RAB7: member RAS oncogene family; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SQSTM1/p62: sequestosome 1

Increased prevalence of granulovacuolar degeneration in C9orf72 mutation.

Granulovacuolar degeneration (GVD) is usually found in Alzheimer's disease (AD) cases or in elderly individuals. Its severity correlates positively with the density of neurofibrillary tangles (NFTs). Mechanisms underlying GVD formation are unknown. We assessed the prevalence and distribution of GVD in cases with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Consecutively autopsied cases with FTLD/ALS-TDP and C9orf72 mutations (FTLD/ALS-C9; N = 29), cases with FTLD/ALS-TDP without C9orf72 mutations (FTLD/ALS-nonC9; N = 46), and age-matched healthy controls (N = 40) were studied. The prevalence of GVD was significantly higher in the FTLD/ALS-C9 cases (26/29 cases) than in the FTLD/ALS-nonC9 cases (15/46 cases; Fisher exact test; p < 2×10-6) or in the control group (12/40 individuals; p < 1×10-6). Average Braak stages and ages of death were not significantly different among the groups. The CA2 sector was most frequently affected in the FTLD/ALS-C9 group, whereas the CA1/subiculum was the most vulnerable area in the other groups. Extension of GVD correlated with the clinical duration of the disease in the FTLD/ALS-C9 cases but not in the FTLD/ALS-nonC9 cases. The GVD-containing neurons frequently had dipeptide repeat (DPR) protein inclusions. GVD granules labeled with antibodies directed against charged multivesicular body protein 2B or casein kinase 1δ were attached to DPR inclusions within GVD. Our results suggest that development of GVD and DPR inclusions is related to common pathogenic mechanisms and that GVD is not only associated with NFTs seen in AD cases or aging individuals.

Transcriptomic analysis under ethylene precursor treatment uncovers the regulation of gene expression linked to sexual reproduction in the dioecious red alga Pyropia pseudolinearis

The marine red algal genus Pyropia, class Bangiophyceae, includes dioecious and monoecious species; however, the molecular mechanisms underlying control of their sexual reproduction are still poorly understood. In the present study, we demonstrated that application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), promoted the formation of spermatangia and parthenosporangia in male and female gametophytes, respectively, of the dioecious species Pyropia pseudolinearis. In addition, we determined expression profiles of ACC-responsive genes in the gametophytes during sexual reproduction using RNA-Seq and quantitative real-time PCR (qRT-PCR). Genes involved in the regulation of cell division and cell wall organization, such as high-mobility group (PpHMG) and glycosyltransferase family (PpGT14), were found to be upregulated in male and female gametophytes treated with ACC. In addition, the relatively rapid ACC-response of the vesicular-trafficking-related genes, flotillin (PpFLOT), charged multivesicular body protein 5 (PpCHMP5), and peptidase family S8 (PpS8) was shown to occur during male and female sexual reproduction. Expression levels of these six genes in the monoecious species P. yezoensis, which are homologs to ACC-responsive genes in P. pseudolinearis, also increased in gametophytes treated with ACC. These findings could provide new insights into the ACC-regulation of the sexual life cycle in Pyropia species.

Assessment of ESCRT Protein CHMP5 Activity on Client Protein Ubiquitination by Immunoprecipitation and Western Blotting.

The charged multivesicular body protein-5 (CHMP5) is a member of the endosomal-sorting complex required for transport (ESCRT) that controls membrane-scission events in eukaryotic cells. Recent studies have revealed novel functions of CHMP5 beyond its role in the ESCRT machinery, notably as a critical nonenzymatic regulator of the ubiquitination and subsequent degradation of proteins in immune cells. Here we describe an immunoprecipitation and western blot methodology for assessing CHMP5 activity on client protein ubiquitination in T lymphocytes.