ABSTRACT
Medical librarians work collaboratively across all units and missions of academic medical centers. One area where librarians can provide key expertise is in the building and maintenance of Research Information Management Systems (RIMS). At Penn State, the RIMS implementation team has included a medical librarian, research administrators and marketing staff from the College of Medicine (CoM) since its inception in 2016. As our peer institutions implemented or expanded their own RIMS systems, the CoM team has responded to their questions regarding details about the Penn State RIMS instance. The goal of this commentary is to describe how the CoM team has worked collaboratively within Penn State to address questions related to research output, with special emphasis on details pertaining to questions from other institutions.
Subject(s)
Librarians , Libraries, Medical , Professional Role , Humans , Libraries, Medical/organization & administration , Biomedical Research/organization & administration , Academic Medical Centers/organization & administrationABSTRACT
PURPOSE: Self-disclosure statements that are informative, rather than apologetic, have been demonstrated to improve listener perceptions of adults who stutter (Byrd et al., 2017). The purpose of the present study is to investigate the benefits of self-disclosure from the perspective of the speaker and to determine whether self-disclosure statement type is associated with self-perceived outcomes of use. METHOD: A total of 156 adults who stutter completed a survey adapted from a previous study investigating affective, behavioral, and cognitive outcomes of voluntary stuttering. Survey responses were analyzed using descriptive and inferential statistics to determine if there was a significant relationship between self-disclosure statement type and self-reported outcomes. Additionally, responses to two open-ended questions relating to timing of self-disclosure and self-disclosure experiences in general were analyzed using reflexive thematic analysis. RESULTS: Self-disclosure was perceived as beneficial in at least one context by 96.8 % of respondents. Respondents who used an informative self-disclosure statement were more likely to agree that self-disclosure increased confidence and were less likely to report attempting to conceal or avoid stuttering than respondents who used an apologetic self-disclosure statement. Themes relating to additional aspects of self-disclosure experiences included personalized use, social connection, acceptance of stuttering, challenging cognitive distortions, communication is easier, self-empowerment, humor, voluntary stuttering, and vulnerability to prejudice. CONCLUSION: Similar to studies investigating the influence of self-disclosure on listener perceptions, informative self-disclosure is associated with greater self-perceived benefits than apologetic self-disclosure for adults who stutter.
Subject(s)
Stuttering , Adult , Humans , Stuttering/psychology , Self Disclosure , Disclosure , Surveys and Questionnaires , CommunicationABSTRACT
PURPOSE: Self-disclosure describes the act of revealing personal information to another person. To date, researchers in the area of stuttering have primarily demonstrated the utility of self-disclosure through analysis of listener perceptions. This study explores the utility of informative self-disclosure use from the perspectives of adults who stutter with experience using this strategy over time and across contexts. METHOD: Twelve adults who stutter discussed their self-disclosure experiences in semistructured interviews. Interview transcripts were analyzed using interpretative phenomenological analysis to produce themes reflecting the most salient aspects of self-disclosure experiences. Credibility was achieved through bracketing, investigator triangulation, and member checking. RESULTS: Four superordinate themes reflecting experiences shared by all 12 participants were generated. Each superordinate theme contained two to three corresponding subthemes. The superordinate themes included cognitive relief, self-empowerment, social connection, and personalization. These findings reflect the positive impact of informative self-disclosure use on communication and quality of life. CONCLUSIONS: Adults who stutter perceive informative self-disclosure to be an effective strategy that provides various benefits to the speaker, in addition to facilitating positive listener perceptions. Clinicians should encourage clients to self-disclose in an informative and personalized manner, provide opportunities for practice, and support clients in determining when and where it is most beneficial for them to implement this strategy.
Subject(s)
Stuttering , Adult , Communication , Disclosure , Humans , Quality of Life/psychology , Stuttering/diagnosis , Stuttering/psychology , Stuttering/therapyABSTRACT
BACKGROUND: Anti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular mechanisms by which this occurs remain poorly understood. Tumor-derived small extracellular vesicles (sEVs) have emerged as critical regulators in modulating the response to immunotherapy. In this study, we investigated the role of neuroblastoma-derived sEVs in promoting resistance to the anti-GD2 monoclonal antibody dinutuximab. Moreover, to determine whether pharmacologic inhibition of sEV secretion sensitizes tumors to dinutuximab treatment, we combined dinutuximab with tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion. METHODS: We investigated the role of neuroblastoma-derived sEVs in modulating the response to dinutuximab by utilizing the syngeneic 9464D-GD2 mouse model. The effect of neuroblastoma-derived sEVs in modulating the tumor microenvironment (TME) and host immune system were evaluated by RNA-sequencing and flow cytometry. Importantly, we used this mouse model to investigate the efficacy of tipifarnib in sensitizing neuroblastoma tumors to dinutuximab. The effect of tipifarnib on both the TME and host immune system were assessed by flow cytometry. RESULTS: We demonstrated that neuroblastoma-derived sEVs significantly attenuated the efficacy of dinutuximab in vivo and modulated tumor immune cell infiltration upon dinutuximab treatment to create an immunosuppressive TME that contains more tumor-associated macrophages and fewer tumor-infiltrating NK cells. In addition, we demonstrated that neuroblastoma-derived sEVs suppress splenic NK cell maturation in vivo and dinutuximab-induced NK cell-mediated antibody-dependent cellular cytotoxicity in vitro. Importantly, tipifarnib drastically enhanced the efficacy of dinutuximab-mediated inhibition of tumor growth and prevented the immunosuppressive effects of neuroblastoma-derived sEVs in vivo. CONCLUSIONS: These preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to dinutuximab and suggest that tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to enhance the antitumor efficacy of anti-GD2 immunotherapy in high-risk neuroblastoma patients.
Subject(s)
Antineoplastic Agents , Extracellular Vesicles , Neuroblastoma , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Humans , Immunologic Factors/therapeutic use , Immunotherapy , Mice , Neuroblastoma/pathology , Quinolones , Tumor MicroenvironmentABSTRACT
Tumor necrosis commonly exists and predicts poor prognoses in many cancers. Although it is thought to result from chronic ischemia, the underlying nature and mechanisms driving the involved cell death remain obscure. Here, we show that necrosis in glioblastoma (GBM) involves neutrophil-triggered ferroptosis. In a hyperactivated transcriptional coactivator with PDZ-binding motif-driven GBM mouse model, neutrophils coincide with necrosis temporally and spatially. Neutrophil depletion dampens necrosis. Neutrophils isolated from mouse brain tumors kill cocultured tumor cells. Mechanistically, neutrophils induce iron-dependent accumulation of lipid peroxides within tumor cells by transferring myeloperoxidase-containing granules into tumor cells. Inhibition or depletion of myeloperoxidase suppresses neutrophil-induced tumor cell cytotoxicity. Intratumoral glutathione peroxidase 4 overexpression or acyl-CoA synthetase long chain family member 4 depletion diminishes necrosis and aggressiveness of tumors. Furthermore, analyses of human GBMs support that neutrophils and ferroptosis are associated with necrosis and predict poor survival. Thus, our study identifies ferroptosis as the underlying nature of necrosis in GBMs and reveals a pro-tumorigenic role of ferroptosis. Together, we propose that certain tumor damage(s) occurring during early tumor progression (i.e. ischemia) recruits neutrophils to the site of tissue damage and thereby results in a positive feedback loop, amplifying GBM necrosis development to its fullest extent.
Subject(s)
Ferroptosis , Glioblastoma/physiopathology , Neutrophils/immunology , Animals , Cell Line, Tumor , Coenzyme A Ligases/genetics , Coenzyme A Ligases/immunology , Disease Progression , Female , Glioblastoma/genetics , Glioblastoma/immunology , Glioblastoma/pathology , Humans , Iron/immunology , Mice , Mice, Nude , Necrosis , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/immunologyABSTRACT
FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.
Subject(s)
Antineoplastic Agents/therapeutic use , Glucocorticoids/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/biosynthesis , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11/biosynthesis , Bcl-2-Like Protein 11/genetics , Benzothiazoles/pharmacology , Benzothiazoles/therapeutic use , Computer Simulation , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Drug Resistance, Neoplasm , Drug Synergism , Gene Expression Regulation, Leukemic/drug effects , Glucocorticoids/pharmacology , Humans , Inflammation/genetics , Mice , Myeloid Cell Leukemia Sequence 1 Protein/biosynthesis , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplastic Stem Cells/drug effects , Phenylurea Compounds/pharmacology , Phenylurea Compounds/therapeutic use , Protein Kinase Inhibitors/pharmacology , Selection, Genetic , Transcriptome , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
FTY720 (fingolimod) is a FDA-approved sphingosine analog that is phosphorylated in vivo to modulate sphingosine-1-phosphate receptor (S1PR) signaling for immunosuppression in patients with refractory multiple sclerosis. FTY720 also exhibits promising anticancer efficacy in several preclinical models. While FTY720-induced cytotoxicity is not due to S1PR signaling, the mechanism remains unclear and is reported to occur through various cell death pathways. Here, we performed a systematic, mechanistic study of FTY720-induced cell death in acute myeloid leukemia (AML). We found that FTY720 induced cell death in a panel of genetically diverse AML cell lines that was accompanied by rapid phosphatidylserine (PS) externalization. Importantly, FTY720-induced PS exposure was not due to any direct effects on plasma membrane integrity and was independent of canonical signaling by regulated cell death pathways known to activate lipid flip-flop, including caspase-dependent apoptosis/pyroptosis, necroptosis, ferroptosis, and reactive oxygen species-mediated cell death. Notably, PS exposure required cellular vacuolization induced by defects in endocytic trafficking and was suppressed by the inhibition of PP2A and shedding of Annexin V-positive subcellular particles. Collectively, our studies reveal a non-canonical pathway underlying PS externalization and cell death in AML to provide mechanistic insight into the antitumor properties of FTY720.
Subject(s)
Apoptosis , Cell Membrane/metabolism , Fingolimod Hydrochloride/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Necroptosis , Phosphatidylserines/metabolism , Caspases/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Sphingosine 1 Phosphate Receptor Modulators/pharmacologyABSTRACT
The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Phagosomes/metabolism , Cells, Cultured , HEK293 Cells , HeLa Cells , HumansABSTRACT
During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.
Subject(s)
Autophagosomes/metabolism , Autophagy-Related Proteins/metabolism , Autophagy , Endoplasmic Reticulum/metabolism , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Amino Acid Sequence , Autophagy-Related Proteins/genetics , HEK293 Cells , Humans , Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphatidylinositol Phosphates/metabolism , Sequence HomologyABSTRACT
Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.
Subject(s)
Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/ultrastructure , Microtubule-Associated Proteins/metabolism , Amino Acid Motifs , Animals , Autophagy , Autophagy-Related Protein 8 Family/metabolism , Carrier Proteins/metabolism , Fluorescence Resonance Energy Transfer/methods , Humans , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy/methods , Microtubule-Associated Proteins/ultrastructure , Mitophagy , Peptides/metabolism , Protein Binding , Protein Interaction Domains and Motifs/physiologyABSTRACT
Macroautophagy (herein referred to as autophagy) is a highly conserved stress response that engulfs damaged proteins, lipids, and/or organelles within double-membrane vesicles called autophagosomes for lysosomal degradation. Dysregulated autophagy is a hallmark of cancer; and thus, there is great interest in modulating autophagy for cancer therapy. Sphingolipids regulate each step of autophagosome biogenesis with roles for sphingolipid metabolites and enzymes spanning from the initial step of de novo ceramide synthesis to the sphingosine-1-phosphate lyase 1-mediated exit from the sphingolipid pathway. Notably, sphingolipid metabolism occurs at several of the organelles that contribute to autophagosome biogenesis to suggest that local changes in sphingolipids may regulate autophagy. As sphingolipid metabolism is frequently dysregulated in cancer, a molecular understanding of sphingolipids in stress-induced autophagy may provide insight into the mechanisms driving tumor development and progression. On the contrary, modulation of sphingolipid metabolites and/or enzymes can induce autophagy-dependent cell death for cancer therapy. This chapter will overview the major steps in mammalian autophagy, discuss the regulation of each step by sphingolipid metabolites, and describe the functions of sphingolipid-mediated autophagy in cancer. While our understanding of the signaling and biophysical properties of sphingolipids in autophagy remains in its infancy, the unique cross talk between the two pathways is an exciting area for further development, particularly in the context of cancer therapy.
Subject(s)
Autophagy , Endocytosis/drug effects , Neoplasms/pathology , Signal Transduction/drug effects , Sphingolipids/pharmacology , Animals , Humans , Neoplasms/drug therapy , Protein TransportABSTRACT
The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.
Subject(s)
Autophagy , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomes/metabolism , Gene Expression Regulation , Lysosomes/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Carrier Proteins , Cell Membrane/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , RNA, Small Interfering/metabolism , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the enzyme that produces it, SPHK1 (sphingosine kinase 1), regulate many processes important for the etiology of cancer. It has been suggested that SPHK1 levels are regulated by the tumor suppressor protein TP53, a key regulator of cell cycle arrest, apoptosis, and macroautophagy/autophagy. However, little is still known of the relationship between TP53 and SPHK1 activity in the regulation of these processes. To explore this link, we examined the effects of inhibiting SPHK1 in wild-type and TP53 null cancer cell lines. SK1-I, an analog of sphingosine and isozyme-specific SPHK1 inhibitor, suppressed cancer cell growth and clonogenic survival in a TP53-dependent manner. It also more strongly enhanced intrinsic apoptosis in wild-type TP53 cells than in isogenic TP53 null cells. Intriguingly, SK1-I induced phosphorylation of TP53 on Ser15, which increases its transcriptional activity. Consequently, levels of TP53 downstream targets such as pro-apoptotic members of the BCL2 family, including BAX, BAK1, and BID were increased in wild-type but not in TP53 null cells. Inhibition of SPHK1 also increased the formation of autophagic and multivesicular bodies, and increased processing of LC3 and its localization within acidic compartments in a TP53-dependent manner. SK1-I also induced massive accumulation of vacuoles, enhanced autophagy, and increased cell death in an SPHK1-dependent manner that also required TP53 expression. Importantly, downregulation of the key regulators of autophagic flux, BECN1 and ATG5, dramatically decreased the cytotoxicity of SK1-I only in cells with TP53 expression. Hence, our results reveal that TP53 plays an important role in vacuole-associated cell death induced by SPHK1 inhibition in cancer cells.
Subject(s)
Apoptosis , Beclin-1/metabolism , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Amino Alcohols/pharmacology , Apoptosis/drug effects , Autophagy , Autophagy-Related Protein 5/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Microtubule-Associated Proteins/metabolism , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Vacuoles/drug effects , Vacuoles/metabolism , Vacuoles/ultrastructureABSTRACT
Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.
Subject(s)
Autophagy-Related Proteins/deficiency , Caspase 8/metabolism , Vesicular Transport Proteins/deficiency , Apoptosis/physiology , Autophagosomes/metabolism , Autophagy/physiology , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Line, Tumor , Enzyme Activation , HeLa Cells , Humans , NF-kappa B/metabolism , Signal Transduction , THP-1 Cells , Transfection , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
Endocytosis, and the subsequent trafficking of endosomes, requires dynamic physical alterations in membrane shape that are mediated in part by endophilin proteins. The endophilin B family of proteins contains an N-terminal Bin/amphiphysin/Rvs (N-BAR) domain that induces membrane curvature to regulate intracellular membrane dynamics. Whereas endophilin B1 (SH3GLB1/Bif-1) is known to be involved in a number of cellular processes, including apoptosis, autophagy, and endocytosis, the cellular function of endophilin B2 (SH3GLB2) is not well understood. In this study, we used genetic approaches that revealed that endophilin B2 is not required for embryonic development in vivo but that endophilin B2 deficiency impairs endosomal trafficking in vitro, as evidenced by suppressed endosome acidification, EGFR degradation, autophagic flux, and influenza A viral RNA nuclear entry and replication. Mechanistically, although the loss of endophilin B2 did not affect endocytic internalization and lysosomal function, endophilin B2 appeared to regulate the trafficking of endocytic vesicles and autophagosomes to late endosomes or lysosomes. Moreover, we also found that despite having an intracellular localization and tissue distribution similar to endophilin B1, endophilin B2 is dispensable for mitochondrial apoptosis. Taken together, our findings suggest that endophilin B2 positively regulates the endocytic pathway in response to growth factor signaling, autophagy induction, and viral entry.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy , Carrier Proteins/metabolism , Endosomes/metabolism , Epidermal Growth Factor/metabolism , ErbB Receptors/agonists , Signal Transduction , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Line , Cells, Cultured , Endocytosis , Endosomes/virology , ErbB Receptors/metabolism , Humans , Influenza A virus/physiology , Mice, Inbred C57BL , Mice, Knockout , Organ Specificity , Organelle Biogenesis , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Virus Internalization , Virus ReplicationABSTRACT
Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1G82D is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis, and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for Sphk1, sphingosine, and autophagic machinery in endocytic membrane trafficking.
Subject(s)
Autophagy , Endocytosis , Endosomes/metabolism , Intracellular Membranes/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Amino Alcohols/pharmacology , Animals , Autophagy/drug effects , Autophagy-Related Protein 5/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Mice , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Sphingolipids/metabolism , Vacuoles/drug effects , Vacuoles/metabolismABSTRACT
Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Proteins/metabolism , Dynamin II/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Autophagy , Cell Proliferation , Cells, Cultured , HeLa Cells , Humans , Mice , Mice, Knockout , NIH 3T3 Cells , Protein TransportABSTRACT
Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eµ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9(+)GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Lymphoma/metabolism , Lymphoma/pathology , Mitophagy , Proto-Oncogene Proteins c-myc/metabolism , Animals , Humans , Mice , Models, BiologicalABSTRACT
Nanotechnologies, while small in size, widen the scope of drug delivery options for compounds with problematic pharmacokinetics, such as bioactive sphingolipids. We describe the development of historical sphingolipid nanotechnologies, such as nanoliposomes, and project future uses for a broad repertoire of nanoscale sphingolipid therapy formulations. In particular, we describe sphingo-nanotherapies for treatment of cancer, inflammatory disease, and cardiovascular disease. We conclude with a discussion of the challenges associated with regulatory approval, scale-up, and development of these nanotechnology therapies for clinical applications.
Subject(s)
Nanotechnology , Sphingolipids/administration & dosage , Cardiovascular Diseases/drug therapy , Humans , Immunity/drug effects , Liposomes , Neoplasms/drug therapyABSTRACT
Malignant transformation by oncogenes requires additional genetic/epigenetic changes to overcome enhanced susceptibility to apoptosis. In the present study, we report that Bif-1 (Sh3glb1), a gene encoding a membrane curvaturedriving endophilin protein, is a haploinsufficient tumor suppressor that plays a key role in the prevention of chromosomal instability and suppresses the acquisition of apoptosis resistance during Myc-driven lymphomagenesis. Although a large portion of Bif-1deficient mice harboring an Eµ-Myc transgene displayed embryonic lethality, allelic loss of Bif-1 dramatically accelerated the onset of Myc-induced lymphoma. At the premalignant stage, hemizygous deletion of Bif-1 resulted in an increase in mitochondrial mass, accumulation of DNA damage, and up-regulation of the antiapoptotic protein Mcl-1. Consistently, allelic loss of Bif-1 suppressed the activation of caspase-3 in Myc-induced lymphoma cells. Moreover, we found that Bif-1 is indispensable for the autophagy-dependent clearance of damaged mitochondria (mitophagy), because loss of Bif-1 resulted in the accumulation of endoplasmic reticulumassociated immature autophagosomes and suppressed the maturation of autophagosomes. The results of the present study indicate that Bif-1 haploinsufficiency attenuates mitophagy and results in the promotion of chromosomal instability, which enables tumor cells to efficiently bypass the oncogenic/metabolic pressures for apoptosis. .