cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.11919600
title: <i>Coxiella</i> effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

description: <i>Coxiella burnetii</i>, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a <i>Coxiella</i> transposon mutant library, we observed that transposon insertions in <i>cbu0626</i> led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the <i>Coxiella</i> vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as <i>Coxiella</i>-containing vacuoles (CCVs). We thus renamed this effector CvpF for <i>Coxiella</i> vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, <i>cvpF</i>::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for <i>Coxiella</i> virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal <i>C. burnetii</i> vacuole biogenesis. <b>Abbreviations:</b> ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: <i>Coxiella</i>-containing vacuole; Cvp: <i>Coxiella</i> vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.
DOI: 10.6084/m9.figshare.11919600.v1
title: <i>Coxiella</i> effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

description: <i>Coxiella burnetii</i>, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a <i>Coxiella</i> transposon mutant library, we observed that transposon insertions in <i>cbu0626</i> led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the <i>Coxiella</i> vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as <i>Coxiella</i>-containing vacuoles (CCVs). We thus renamed this effector CvpF for <i>Coxiella</i> vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, <i>cvpF</i>::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for <i>Coxiella</i> virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal <i>C. burnetii</i> vacuole biogenesis. <b>Abbreviations:</b> ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: <i>Coxiella</i>-containing vacuole; Cvp: <i>Coxiella</i> vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.
DOI: 10.6084/m9.figshare.11840955.v2
title: Mechanistic dissection of macro- and micronucleophagy

description: Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes. <b>Abbreviations:</b> DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.
DOI: 10.6084/m9.figshare.11871186
title: The neurosteroid allopregnanolone protects retinal neurons by effects on autophagy and GABRs/GABA<sub>A</sub> receptors in rat glaucoma models

description: In an <i>ex vivo</i> rat glaucoma model using dissected retinas, the neurosteroid allopregnanolone (AlloP) protects retinal ganglion cells (RGCs) via GABR/GABA<sub>A</sub> receptors. To determine the involvement of macroautophagy/autophagy in neuroprotection by AlloP, we examined the effects of autophagy activators, rapamycin and torin 2, and autophagy inhibitors, bafilomycin A<sub>1</sub> and SAR405, on retinal retinal morphology and expression of MAP1 LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) and SQSTM1 (sequestosome 1). Administration of rapamycin or torin 2 exerted partial histological neuroprotection, while combined administration of AlloP with bafilomycin A<sub>1</sub> or SAR405 induced severe degeneration in a hyperbaric condition. Electron microscopic analyses showed that the addition of AlloP significantly increased autophagosomes and degenerative autophagic vacuoles in the retinal nerve fiber layer. Immunoblotting showed that the addition of AlloP or autophagic activators increased the lipidated form of LC3B (LC3B-II) and suppressed SQSTM1. Moreover, bafilomycin A<sub>1</sub> increased LC3B-II and SQSTM1 protein levels in the presence of AlloP without changes in corresponding mRNAs compared to AlloP-treated retinas in a hyperbaric condition. These data indicate that AlloP likely induces a protective form of autophagy in this model. In an <i>in vivo</i> rat model of glaucoma, we also observed neuroprotective effects of AlloP. Injection of polystyrene microbeads into the anterior chamber increased intraocular pressure about 3-fold and induced RGC apoptosis. A single intravitreal injection of AlloP or autophagy activators prevented apoptosis and protected RGCs with autophagy activation. We conclude that AlloP may serve as a potential therapeutic agent for the treatment of glaucoma via diverse mechanisms. <b>Abbreviations:</b> 2HBCD: 2-Hydroxypropyl)-β-cyclodextrin; 3-MA: 3-methyladenine; AlloP: allopregnanolone; AP: autophagosome; AVd: degradative autophagic vacuoles; GCL: ganglion cell layer; INL: inner nuclear layer; IOP: intraocular pressure; IPL: inner plexiform layer; LC3B-I: cytosolic form of LC3B; LCB-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; NDS: neuronal damage score; NFL: nerve fiber layer; OH: ocular hypertension; ON: optic nerve; ONL: outer nuclear layer; OPL: outer plexiform layer; p-STR: scotopic threshold response; RGC: retinal ganglion cells; RT-PCR: real-time reverse transcription polymerase chain reaction; SQSTM1: sequestosome 1; TUNEL: TdT-mediated dUTP Nick End Labeling
DOI: 10.6084/m9.figshare.11840955
title: Mechanistic dissection of macro- and micronucleophagy

description: Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes. <b>Abbreviations:</b> DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.
DOI: 10.6084/m9.figshare.11840955.v1
title: Mechanistic dissection of macro- and micronucleophagy

description: Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes. <b>Abbreviations:</b> DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.
DOI: 10.6084/m9.figshare.11980305
title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

description: Lysosomal Ca<sup>2+</sup> contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca<sup>2+</sup> channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca<sup>2+</sup> levels. The Ca<sup>2+</sup> efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca<sup>2+</sup> level, in which ITPR-independent regulation of Ca<sup>2+</sup> status was observed. Further, TMBIM6 regulated the local release of Ca<sup>2+</sup> through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca<sup>2+</sup> efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca<sup>2+</sup> and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved <i>Tmbim6</i><sup>+/+</sup> mice compared with that in the counter <i>tmbim6</i><sup>−/-</sup> mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca<sup>2+</sup> release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca<sup>2+</sup> modulator that coordinates with autophagy to alleviate metabolism stress.<b>Abbreviations</b>: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6
DOI: 10.6084/m9.figshare.11980305.v1
title: TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

description: Lysosomal Ca<sup>2+</sup> contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca<sup>2+</sup> channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca<sup>2+</sup> levels. The Ca<sup>2+</sup> efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca<sup>2+</sup> level, in which ITPR-independent regulation of Ca<sup>2+</sup> status was observed. Further, TMBIM6 regulated the local release of Ca<sup>2+</sup> through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca<sup>2+</sup> efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca<sup>2+</sup> and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved <i>Tmbim6</i><sup>+/+</sup> mice compared with that in the counter <i>tmbim6</i><sup>−/-</sup> mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca<sup>2+</sup> release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca<sup>2+</sup> modulator that coordinates with autophagy to alleviate metabolism stress.<b>Abbreviations</b>: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6
DOI: 10.6084/m9.figshare.11798499.v2
title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

description: Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis. CMA modulates proteomic organization through selective protein degradation, with targets including metabolic enzymes, cell growth regulators, and neurodegeneration-related proteins. CMA activity is low in <i>ad libitum</i>-fed rodents but is increased by prolonged fasting. AKT negatively regulates CMA at the lysosomal membrane by phosphorylating and inhibiting the CMA regulator GFAP. We have previously reported that long-lived <i>Pou1f1/Pit1</i> mutant (Snell) mice and <i>ghr</i> (growth hormone receptor) knockout mice (<i>ghr</i> KO) have lower AKT activity when fed compared to littermate controls, suggesting the hypothesis that these mice have increased baseline CMA activity. Here, we report that liver lysosomes from fed Snell dwarf mice and <i>ghr</i> KO mice have decreased GFAP phosphorylation and increased CMA substrate uptake activity. Liver lysosomes isolated from fed Snell dwarf mice and <i>ghr</i> KO mice injected with the protease inhibitor leupeptin had increased accumulation of endogenous CMA substrates, compared to littermate controls, suggesting an increase in CMA <i>in vivo</i>. Mice with liver-specific ablation of GH (growth hormone) signaling did not have increased liver CMA, suggesting that a signaling effect resulting from a loss of growth hormone in another tissue causes enhanced CMA in Snell dwarf and <i>ghr</i> KO mice. Finally, we find Snell dwarf mice have decreased protein levels (in liver and kidney) of CIP2A, a well-characterized CMA target protein, without an associated change in <i>Cip2a</i> mRNA. Collectively, these data suggest that CMA is enhanced downstream of an endocrine change resulting from whole-body ablation of GH signaling. <b>Abbreviations:</b> CMA: chaperone-mediated autophagy; GH: growth hormone; <i>ghr</i> KO: growth hormone receptor knockout; LAMP2A: splice variant 1 of <i>Lamp2</i> transcript; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; Li<i>-ghr</i> KO: liver-specific <i>ghr</i> knockout; MA: macroautophagy; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; PBS: phosphate-buffered saline.
DOI: 10.6084/m9.figshare.11880837
title: Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release

description: Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4B<sup>C74A</sup>). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4B<sup>C74A</sup>, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. <b>Abbreviations:</b> ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short