Fusion Of Lysosomes Research Articles

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a–VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome–lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.

Prevention of D-GalN/LPS-induced ALI by 18\u03b2-glycyrrhetinic acid through PXR-mediated inhibition of autophagy degradation

Acute liver injury (ALI) has multiple causes and results in liver dysfunction. Severe or persistent liver injury eventually leads to liver failure and even death. Pregnane X receptor (PXR)-null mice present more severe liver damage and lower rates of autophagy. 18β-glycyrrhetinic acid (GA) has been proposed as a promising hepatoprotective agent. We hypothesized that GA significantly alleivates D-GalN/LPS-induced ALI, which involved in PXR-mediated autophagy and lysosome biogenesis. We found that GA can significantly decrease hepatocyte apoptosis and increase the hepatic autophagy marker LC3-B. Ad-mCherry-GFP-LC3 tandem fluorescence, RNA-seq and real-time PCR indicated that GA may stabilize autophagosomes and lysosomes and inhibit autophagosome–lysosome fusion. Simultaneously, GA markedly activates PXR, even reversing the D-GalN/LPS-induced reduction of PXR and its downstream genes. In contrast, GA has a weak protective effect in pharmacological inhibition of PXR and PXR-null mice, which significantly affected apoptosis- and autophagy-related genes. PXR knockout interferes with the stability of autophagosomes and lysosomes, preventing GA reducing the expression of lysosomal genes such as Cst B and TPP1, and suppressing autophagy flow. Therefore, we believe that GA increases autophagy by inhibiting autophagosome–lysosome fusion and blocked autophagy flux via activation of PXR. In conclusion, our results show that GA activates PXR to regulate autophagy and lysosome biogenesis, represented by inhibiting autophagosome–lysosome fusion and stabilization of lysosome. These results identify a new mechanism by which GA-dependent PXR activation reduces D-GalN/LPS-induced acute liver injury.

P27 controls autophagic vesicle trafficking in glucose-deprived cells via the regulation of ATAT1-mediated microtubule acetylation

The cyclin-dependent kinase inhibitor p27Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome–lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27−/− MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27+/+ MEFs and impaired autophagy flux, similar to that observed in p27−/− cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism.

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.

Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington\u2019s disease

Huntington’s disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Puerarin Restores Autophagosome-Lysosome Fusion to Alleviate Cadmium-Induced Autophagy Blockade via Restoring the Expression of Rab7 in Hepatocytes.

Autophagic dysfunction is one of the main mechanisms by which the environmental pollutant cadmium (Cd) induces cell injury. Puerarin (Pue, a monomeric Chinese herbal medicine extract) has been reported to alleviate Cd-induced cell injury by regulating autophagy pathways; however, its detailed mechanisms are unclear. In the present study, to investigate the detailed mechanisms by which Pue targets autophagy to alleviate Cd hepatotoxicity, alpha mouse liver 12 (AML12) cells were used to construct a model of Cd-induced hepatocyte injury in vitro. First, the protective effect of Pue on Cd-induced cell injury was confirmed by changes in cell proliferation, cell morphology, and cell ultrastructure. Next, we found that Pue activated autophagy and mitigated Cd-induced autophagy blockade. In this process, the lysosome was further activated and the lysosomal degradation capacity was strengthened. We also found that Pue restored the autophagosome-lysosome fusion and the expression of Rab7 in Cd-exposed hepatocytes. However, the fusion of autophagosomes with lysosomes and autophagic flux were inhibited after knocking down Rab7, and were further inhibited after combined treatment with Cd. In addition, after knocking down Rab7, the protective effects of Pue on restoring autophagosome-lysosome fusion and alleviating autophagy blockade in Cd-exposed cells were inhibited. In conclusion, Pue-mediated alleviation of Cd-induced hepatocyte injury was related to the activation of autophagy and the alleviation of autophagy blockade. Pue also restored the fusion of autophagosomes and lysosomes by restoring the protein expression of Rab7, thereby alleviating Cd-induced autophagy blockade in hepatocytes.

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1‐dependent TFEB/TFE3 regulation

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662*;VPS41c.1423‐2A>G and VPS41R662*) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41‐depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson’s disease, co‐expression of VPS41S285P/VPS41R662* abolished the neuroprotective function of VPS41 against α‐synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

Metformin Attenuates Monosodium-Iodoacetate-Induced Osteoarthritis via Regulation of Pain Mediators and the Autophagy-Lysosomal Pathway.

Osteoarthritis (OA) is the most common degenerative arthritis associated with pain and cartilage destruction in the elderly; it is known to be involved in inflammation as well. A drug called celecoxib is commonly used in patients with osteoarthritis to control pain. Metformin is used to treat type 2 diabetes but also exhibits regulation of the autophagy pathway. The purpose of this study is to investigate whether metformin can treat monosodium iodoacetate (MIA)-induced OA in rats. Metformin was administered orally every day to rats with OA. Paw-withdrawal latency and threshold were used to assess pain severity. Cartilage damage and pain mediators in dorsal root ganglia were evaluated by histological analysis and a scoring system. Relative mRNA expression was measured by real-time PCR. Metformin reduced the progression of experimental OA and showed both antinociceptive properties and cartilage protection. The combined administration of metformin and celecoxib controlled cartilage damage more effectively than metformin alone. In chondrocytes from OA patients, metformin reduced catabolic factor gene expression and inflammatory cell death factor expression, increased LC3Ⅱb, p62, and LAMP1 expression, and induced an autophagy–lysosome fusion phenotype. We investigated if metformin treatment reduces cartilage damage and inflammatory cell death of chondrocytes. The results suggest the potential for the therapeutic use of metformin in OA patients based on its ability to suppress pain and protect cartilage.

POSTN promotes diabetic vascular calcification by interfering with autophagic flux

Autophagy is a lysosomal degradative process that is closely related to the pathogenesis of vascular calcification. Recent evidence suggests that periostin (POSTN) is a unique extracellular matrix protein that is associated with diabetic vascular complications. The aim of current study is to investigate the role of POSTN in diabetic vascular calcification and the underlying mechanisms. Results showed that POSTN was highly upregulated in both calcified arteries of diabetic rats and AGEs-BSA mediated vascular smooth muscle cell (VSMC) calcification. POSTN blocked autophagic flux during the diabetic calcification process, as evidenced by increased protein expression of Beclin1, LC3-II, and P62, as well as the co-localization of LC3-II and LAMP1. Inhibition of POSTN alleviated AGEs-BSA-induced autophagic flux blockade, thereby attenuating AGEs-BSA-induced VSMC calcification. Mechanistically, the upregulation of POSTN impaired the fusion of autophagosomes and lysosome and resulted in the autophagic flux blockade in AGEs-BSA-treated VSMC. Furthermore, this autophagic blockade was intracellular ROS-dependent. In summary, this study uncovered a novel mechanism of POSTN in autophagy regulation of diabetic vascular calcification.

The lysosomal membrane protein Sidt2 is a vital regulator of mitochondrial quality control in skeletal muscle.

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

Mild hypothermia pretreatment extenuates liver ischemia-reperfusion injury through Rab7-mediated autophagosomes-lysosomes fusion

Liver ischemia-reperfusion (IR) injury is an unavoidable pathological process in transplantation, closely related to poor prognosis. To date, there has been no clear therapeutic measure. We previously reported that mild hypothermia (MH), a widely used therapy, can exert significant protective effect against liver IR injury. Among the multiple mechanisms underlying the therapeutic effect of MH, autophagy flux drew our special attention. In this study, we evaluated the role of autophagy flux in IR injury and thereby explored the relationship between MH and autophagy flux in IR injury. We developed in vivo and in vitro models for hepatic IR injury. By autophagy flux assay with Western blotting and immunofluorescence, we found that MH restricts heavy accumulation of autophagosomes (APs) during IR injury. Activation and blocking of the autophagy flux unraveled that accumulation of APs further aggravated IR injury. Further, MH reduces APs accumulation to restore autophagy flux by regulating the fusion of APs and lysosomes. Besides, MH upregulated the level of Rab7 protein expression that was seriously impaired during IR injury. Inhibition of Rab7 expression increased apoptosis of liver cells and reduced the degree of overlap between APs and lysosomes. The results were reversed upon activation of Rab7. In conclusion, MH can alleviate liver IR injury by regulating the Rab7-mediated APs-lysosomes fusion that reduces APs accumulation. This can provide a theoretical basis for the further application of MH in related clinical diseases.

72399 Epigenetic Modification of Macrophages Contribute to Protective Memory in Against Staphylococcus aureus

ABSTRACT IMPACT: This work may provide new targets for vaccine and immunotherapeutic development against MRSA infections. OBJECTIVES/GOALS: Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Patients with SA SSSI have a high 1-year recurrence. We have shown innate memory protects mice against SA SSSI. The goal of this project is to determine epigenetic mechanisms of protective memory against SA SSSI. METHODS/STUDY POPULATION: We have shown macrophages (Mf) afford protective memory against recurrent SA SSSI in mice. Priming by prior infection reduced skin lesion size and MRSA burden, which correlated with increased Mf in abscesses and lymph nodes. Priming potentiated the opsonophagocytic killing of SA by bone-marrow derived Mf (BMDM) in vitro, and their adoptive transfer into naive skin afforded protective efficacy in vivo. Here, we investigated epigenetic mechanisms of anti-SA efficacy in BMDMs. BMDM from naive (uninfected) or primed (SA SSSI) wild-type C57Bl/6 mice were cultured ex vivo. DNA from BMDM groups were isolated and analyzed for methylation changes using reduced representation bisulfite sequencing (RRBS). Pathway analyses of methylation changes were determined with Panther. RESULTS/ANTICIPATED RESULTS: Present findings indicate the protective memory afforded by BMDM was mediated by epigenetic modifications of the DNA. Using RRBS, we profiled differentially methylated regions (DMR) in DNA from naive vs. primed BMDM. Primed BMDM exhibited significantly different DMRs as compared to naive BMDM. Proximity to known genes were mapped using GREAT. Pathway analyses revealed DMRs predominant in genes integral to immune modulation, such as integrin signaling, cytokine/chemokine networks, and growth regulation. For example, SA-primed BMDM were hypermethylated proximate to GIMAP8 versus naive BMDM, suggesting repression of this protein. Gimap family ligands are small GTPase immune-associated proteins expressed in immune cells known to regulate macrophage lysosomal fusion during parasite infection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These findings reveal epigenetic mechanisms of macrophage innate memory against recurrent MRSA infection. Functional testing of these genes in response to SA infection is needed to confirm their protective role. These insights may provide new targets for vaccine and immunotherapeutic development against MRSA.

Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium.

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1–30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.

Lysosomes and Cancer Progression: A Malignant Liaison.

During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.

Overcoming Chemoresistance: Altering pH of Cellular Compartments by Chloroquine and Hydroxychloroquine.

Resistance to the anti-cancer effects of chemotherapeutic agents (chemoresistance) is a major issue for people living with cancer and their providers. A diverse set of cellular and inter-organellar signaling changes have been implicated in chemoresistance, but it is still unclear what processes lead to chemoresistance and effective strategies to overcome chemoresistance are lacking. The anti-malaria drugs, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are being used for the treatment of various cancers and CQ and HCQ are used in combination with chemotherapeutic drugs to enhance their anti-cancer effects. The widely accepted anti-cancer effect of CQ and HCQ is their ability to inhibit autophagic flux. As diprotic weak bases, CQ and HCQ preferentially accumulate in acidic organelles and neutralize their luminal pH. In addition, CQ and HCQ acidify the cytosolic and extracellular environments; processes implicated in tumorigenesis and cancer. Thus, the anti-cancer effects of CQ and HCQ extend beyond autophagy inhibition. The present review summarizes effects of CQ, HCQ and proton pump inhibitors on pH of various cellular compartments and discuss potential mechanisms underlying their pH-dependent anti-cancer effects. The mechanisms considered here include their ability to de-acidify lysosomes and inhibit autophagosome lysosome fusion, to de-acidify Golgi apparatus and secretory vesicles thus affecting secretion, and to acidify cytoplasm thus disturbing aerobic metabolism. Further, we review the ability of these agents to prevent chemotherapeutic drugs from accumulating in acidic organelles and altering their cytosolic concentrations.

Activity-Based Fluorescent Molecular Logic Gate Probe for Dynamic Tracking of Mitophagy Induced by Oxidative Stress.

Visualizing and modulating the mitophagy process is essential for understanding the role of mitophagy in cellular homeostasis, physiology, and pathology. To overcome the sensing limitation of available mitophagy probes to only lysosome fusion or degradation, a molecular logic gate probe showing multiple fluorescence responses to different mitophagy stages was proposed in this study to sense the oxidative stress-induced mitophagy via a dual-channel mode. This new fluorescent molecular logic gate probe, Mito-PN, was composed by integrating a peroxynitrite-responsive 1,8-naphthalimide with an acidity-activatable rhodamine spirolactam and possesses the mitochondria-targeting capability due to its triphenylphosphonium group. This probe is able to sense both the mitophagy initiation triggered by peroxynitrite and lysosome fusion at different fluorescence wavelengths. It can be rapidly activated by mitochondrial peroxynitrite to turn on the green fluorescence of naphthalimide, and subsequent lysosome/mitophagosome fusion activates the probe with protons to generate red fluorescence. Moreover, our preliminary results demonstrate that the fluorescence response of Mito-PN to peroxynitrite-induced mitophagy can be discriminated from the mitophagy stimulated by carbonyl cyanide m-chlorophenyl hydrazone, which further proves the specific mitophagy tracking ability of Mito-PN. Overall, this research offers a potentially powerful tool for studying the role played by peroxynitrite in mitophagy and provides a versatile strategy for monitoring oxidative stress-related pathological processes.

Expanding the Spectrum of AP5Z1‐Related Hereditary Spastic Paraplegia (HSP‐SPG48): A Multicenter Study on a Rare Disease

Expanding the Spectrum of <scp><i>AP5Z1‐</i></scp>Related Hereditary Spastic Paraplegia (<scp>HSP‐SPG48</scp>): A Multicenter Study on a Rare Disease

Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance

Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin–paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome–lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum–taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum–taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.

Translation of Mycobacterium Survival Strategy to Develop a Lipo‐peptide based Fusion Inhibitor**

AbstractThe entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad‐spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome–lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40‐repeat protein suggest that the trp‐asp (WD) sequence is placed at distorted β‐meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo‐peptide sequence (myr‐WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo‐dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad‐spectrum antiviral agent.

SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan

Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson’s disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal–lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.