cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.13300726
title: A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway

description: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (Ub<sup>G76V</sup>-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both Ub<sup>G76V</sup>-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with <i>Zfp418</i> overexpression in HEK293 cells, we evaluated <i>Zfp418</i> knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from <i>Zfp418</i> knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of <i>Dapk2</i> and <i>Fyco1</i>, which are involved in autophagy. RNA-seq from <i>Zfp418</i> knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function. <b>Abbreviations:</b> ACTN2, actinin alpha 2; ALP, autophagy-lysosomal pathway; COPB1, COPI coat complex subunit beta 1; DAPK2, death associated protein kinase 2; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; HEK293, human embryonic kidney cells 293; HTS, high-throughput screen; LC3, microtubule associated protein 1 light chain 3; NRVMs, neonatal rat ventricular myocytes; RNA-seq, RNA sequencing; RPS6, ribosomal protein S6; TNNI3, troponin I, cardiac 3; UPS, ubiquitin-proteasome system; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; VPS28, VPS28 subunit of ESCRT-I; ZNF418/ZFP418, zinc finger protein 418.
DOI: 10.6084/m9.figshare.13220222.v3
title: HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1

description: Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing world health problem with a more favorable prognosis for patients with human papillomavirus (HPV)-positive tumors compared to those with HPV-negative OPSCC. How HPV confers a less aggressive phenotype, however, remains undefined. We demonstrated that HPV-positive OPSCC cells display reduced macroautophagy/autophagy activity, mediated by the ability of HPV-E7 to interact with AMBRA1, to compete with its binding to BECN1 and to trigger its calpain-dependent degradation. Moreover, we have shown that AMBRA1 downregulation and pharmacological inhibition of autophagy sensitized HPV-negative OPSCC cells to the cytotoxic effects of cisplatin. Importantly, semi-quantitative immunohistochemical analysis in primary OPSCCs confirmed that AMBRA1 expression is reduced in HPV-positive compared to HPV-negative tumors. Collectively, these data identify AMBRA1 as a key target of HPV to impair autophagy and propose the targeting of autophagy as a viable therapeutic strategy to improve treatment response of HPV-negative OPSCC. <b>Abbreviations</b>: AMBRA1: autophagy and beclin 1 regulator 1; CDDP: cisplatin (CDDP); FFPE: formalin-fixed paraffin-embedded (FFPE); HNC: head and neck cancers (HNC); HPV: human papillomavirus (HPV); hrHPV: high risk human papillomavirus (hrHPV); OCSCC: oral cavity squamous carcinomas (OCSSC); OPSCC: oropharyngeal squamous cell carcinoma (OPSCC); OS: overall survival (OS); qPCR: quantitative polymerase chain reaction; RB1: RB transcriptional corepressor 1; ROC: receiver operating characteristic curve (ROC).
DOI: 10.6084/m9.figshare.13300726.v1
title: A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway

description: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (Ub<sup>G76V</sup>-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both Ub<sup>G76V</sup>-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with <i>Zfp418</i> overexpression in HEK293 cells, we evaluated <i>Zfp418</i> knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from <i>Zfp418</i> knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of <i>Dapk2</i> and <i>Fyco1</i>, which are involved in autophagy. RNA-seq from <i>Zfp418</i> knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function. <b>Abbreviations:</b> ACTN2, actinin alpha 2; ALP, autophagy-lysosomal pathway; COPB1, COPI coat complex subunit beta 1; DAPK2, death associated protein kinase 2; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; HEK293, human embryonic kidney cells 293; HTS, high-throughput screen; LC3, microtubule associated protein 1 light chain 3; NRVMs, neonatal rat ventricular myocytes; RNA-seq, RNA sequencing; RPS6, ribosomal protein S6; TNNI3, troponin I, cardiac 3; UPS, ubiquitin-proteasome system; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; VPS28, VPS28 subunit of ESCRT-I; ZNF418/ZFP418, zinc finger protein 418.
DOI: 10.6084/m9.figshare.13220222
title: HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1

description: Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing world health problem with a more favorable prognosis for patients with human papillomavirus (HPV)-positive tumors compared to those with HPV-negative OPSCC. How HPV confers a less aggressive phenotype, however, remains undefined. We demonstrated that HPV-positive OPSCC cells display reduced macroautophagy/autophagy activity, mediated by the ability of HPV-E7 to interact with AMBRA1, to compete with its binding to BECN1 and to trigger its calpain-dependent degradation. Moreover, we have shown that AMBRA1 downregulation and pharmacological inhibition of autophagy sensitized HPV-negative OPSCC cells to the cytotoxic effects of cisplatin. Importantly, semi-quantitative immunohistochemical analysis in primary OPSCCs confirmed that AMBRA1 expression is reduced in HPV-positive compared to HPV-negative tumors. Collectively, these data identify AMBRA1 as a key target of HPV to impair autophagy and propose the targeting of autophagy as a viable therapeutic strategy to improve treatment response of HPV-negative OPSCC. <b>Abbreviations</b>: AMBRA1: autophagy and beclin 1 regulator 1; CDDP: cisplatin (CDDP); FFPE: formalin-fixed paraffin-embedded (FFPE); HNC: head and neck cancers (HNC); HPV: human papillomavirus (HPV); hrHPV: high risk human papillomavirus (hrHPV); OCSCC: oral cavity squamous carcinomas (OCSSC); OPSCC: oropharyngeal squamous cell carcinoma (OPSCC); OS: overall survival (OS); qPCR: quantitative polymerase chain reaction; RB1: RB transcriptional corepressor 1; ROC: receiver operating characteristic curve (ROC).
DOI: 10.6084/m9.figshare.13264752
title: Lysosomal targeting of autophagosomes by the TECPR domain of TECPR2

description: TECPR2 (tectonin beta-propeller repeat containing 2) is a large, multi-domain protein comprised of an amino-terminal WD domain, a middle unstructured region and a carboxy-terminal TEPCR domain comprises of six TECPR repeats followed by a functional LIR motif. Human <i>TECPR2</i> mutations are linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder. Here we show that basal macroautophagic/autophagic flux is impaired in SPG49 patient fibroblasts in the form of accumulated autophagosomes. Ectopic expression of either full length TECPR2 or the TECPR domain rescued autophagy in patient fibroblasts in a LIR-dependent manner. Moreover, this domain is recruited to the cytosolic leaflet of autophagosomal and lysosomal membranes in a LIR- and VAMP8-dependent manner, respectively. These findings provide evidence for a new role of the TECPR domain in particular, and TECPR2 in general, in lysosomal targeting of autophagosomes <i>via</i> association with Atg8-family proteins on autophagosomes and VAMP8 on lysosomes. <b>Abbreviations:</b> HOPS: homotypic fusion and vacuole protein sorting; LIR: LC3-interacting region; SPG49: spastic paraplegia type 49; STX17: syntaxin 17; TECPR2: tectonin beta-propeller repeat containing 2; VAMP8: vesicle associated membrane protein 8
DOI: 10.6084/m9.figshare.13264752.v3
title: Lysosomal targeting of autophagosomes by the TECPR domain of TECPR2

description: TECPR2 (tectonin beta-propeller repeat containing 2) is a large, multi-domain protein comprised of an amino-terminal WD domain, a middle unstructured region and a carboxy-terminal TEPCR domain comprises of six TECPR repeats followed by a functional LIR motif. Human <i>TECPR2</i> mutations are linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder. Here we show that basal macroautophagic/autophagic flux is impaired in SPG49 patient fibroblasts in the form of accumulated autophagosomes. Ectopic expression of either full length TECPR2 or the TECPR domain rescued autophagy in patient fibroblasts in a LIR-dependent manner. Moreover, this domain is recruited to the cytosolic leaflet of autophagosomal and lysosomal membranes in a LIR- and VAMP8-dependent manner, respectively. These findings provide evidence for a new role of the TECPR domain in particular, and TECPR2 in general, in lysosomal targeting of autophagosomes <i>via</i> association with Atg8-family proteins on autophagosomes and VAMP8 on lysosomes. <b>Abbreviations:</b> HOPS: homotypic fusion and vacuole protein sorting; LIR: LC3-interacting region; SPG49: spastic paraplegia type 49; STX17: syntaxin 17; TECPR2: tectonin beta-propeller repeat containing 2; VAMP8: vesicle associated membrane protein 8
DOI: 10.6084/m9.figshare.13220222.v2
title: HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1

description: Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing world health problem with a more favorable prognosis for patients with human papillomavirus (HPV)-positive tumors compared to those with HPV-negative OPSCC. How HPV confers a less aggressive phenotype, however, remains undefined. We demonstrated that HPV-positive OPSCC cells display reduced macroautophagy/autophagy activity, mediated by the ability of HPV-E7 to interact with AMBRA1, to compete with its binding to BECN1 and to trigger its calpain-dependent degradation. Moreover, we have shown that AMBRA1 downregulation and pharmacological inhibition of autophagy sensitized HPV-negative OPSCC cells to the cytotoxic effects of cisplatin. Importantly, semi-quantitative immunohistochemical analysis in primary OPSCCs confirmed that AMBRA1 expression is reduced in HPV-positive compared to HPV-negative tumors. Collectively, these data identify AMBRA1 as a key target of HPV to impair autophagy and propose the targeting of autophagy as a viable therapeutic strategy to improve treatment response of HPV-negative OPSCC. <b>Abbreviations</b>: AMBRA1: autophagy and beclin 1 regulator 1; CDDP: cisplatin (CDDP); FFPE: formalin-fixed paraffin-embedded (FFPE); HNC: head and neck cancers (HNC); HPV: human papillomavirus (HPV); hrHPV: high risk human papillomavirus (hrHPV); OCSCC: oral cavity squamous carcinomas (OCSSC); OPSCC: oropharyngeal squamous cell carcinoma (OPSCC); OS: overall survival (OS); qPCR: quantitative polymerase chain reaction; RB1: RB transcriptional corepressor 1; ROC: receiver operating characteristic curve (ROC).
DOI: 10.6084/m9.figshare.13264752.v2
title: Lysosomal targeting of autophagosomes by the TECPR domain of TECPR2

description: TECPR2 (tectonin beta-propeller repeat containing 2) is a large, multi-domain protein comprised of an amino-terminal WD domain, a middle unstructured region and a carboxy-terminal TEPCR domain comprises of six TECPR repeats followed by a functional LIR motif. Human <i>TECPR2</i> mutations are linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder. Here we show that basal macroautophagic/autophagic flux is impaired in SPG49 patient fibroblasts in the form of accumulated autophagosomes. Ectopic expression of either full length TECPR2 or the TECPR domain rescued autophagy in patient fibroblasts in a LIR-dependent manner. Moreover, this domain is recruited to the cytosolic leaflet of autophagosomal and lysosomal membranes in a LIR- and VAMP8-dependent manner, respectively. These findings provide evidence for a new role of the TECPR domain in particular, and TECPR2 in general, in lysosomal targeting of autophagosomes <i>via</i> association with Atg8-family proteins on autophagosomes and VAMP8 on lysosomes. <b>Abbreviations:</b> HOPS: homotypic fusion and vacuole protein sorting; LIR: LC3-interacting region; SPG49: spastic paraplegia type 49; STX17: syntaxin 17; TECPR2: tectonin beta-propeller repeat containing 2; VAMP8: vesicle associated membrane protein 8
DOI: 10.6084/m9.figshare.13168583
title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein

description: Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1. <b>Abbreviations:</b> AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; Baf: bafilomycin A<sub>1</sub>; BiFC: biomolecular fluorescence complementation; CAMKK2/CAMKKB: calcium/calmodulin dependent protein kinase kinase 2; CC: compound C; DARTS: drug affinity responsive target stability; HUVECs: human umbilical vein endothelial cells; Inda: indatraline; STK11/LKB1: serine/threonine kinase 11; MAPT: microtubule associated protein tau; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; 3-MA: 3-methyladenine; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; Rapa: rapamycin; Sert: sertraline; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; SLC6A4/SERT1: solute carrier family 6 member 4; TFEB: transcription factor EB; VDAC1: voltage dependent anion channel 1; WT: wild-type; WM: wortmannin.
DOI: 10.6084/m9.figshare.13168583.v3
title: Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein

description: Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1. <b>Abbreviations:</b> AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; Baf: bafilomycin A<sub>1</sub>; BiFC: biomolecular fluorescence complementation; CAMKK2/CAMKKB: calcium/calmodulin dependent protein kinase kinase 2; CC: compound C; DARTS: drug affinity responsive target stability; HUVECs: human umbilical vein endothelial cells; Inda: indatraline; STK11/LKB1: serine/threonine kinase 11; MAPT: microtubule associated protein tau; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; 3-MA: 3-methyladenine; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; Rapa: rapamycin; Sert: sertraline; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; SLC6A4/SERT1: solute carrier family 6 member 4; TFEB: transcription factor EB; VDAC1: voltage dependent anion channel 1; WT: wild-type; WM: wortmannin.
DOI: 10.6084/m9.figshare.13365290
title: The conformational and mutational landscape of the ubiquitin-like marker for autophagosome formation in cancer

description: Macroautophagy/autophagy is a cellular process to recycle damaged cellular components, and its modulation can be exploited for disease treatments. A key autophagy player is the ubiquitin-like protein MAP1LC3B/LC3B. Mutations and changes in <i>MAP1LC3B</i> expression occur in cancer samples. However, the investigation of the effects of these mutations on MAP1LC3B protein structure is still missing. Despite many LC3B structures that have been solved, a comprehensive study, including dynamics, has not yet been undertaken. To address this knowledge gap, we assessed nine physical models for biomolecular simulations for their capabilities to describe the structural ensemble of MAP1LC3B. With the resulting MAP1LC3B structural ensembles, we characterized the impact of 26 missense mutations from pan-cancer studies with different approaches, and we experimentally validated our prediction for six variants using cellular assays. Our findings shed light on damaging or neutral mutations in MAP1LC3B, providing an atlas of its modifications in cancer. In particular, P32Q mutation was found detrimental for protein stability with a propensity to aggregation. In a broader context, our framework can be applied to assess the pathogenicity of protein mutations or to prioritize variants for experimental studies, allowing to comprehensively account for different aspects that mutational events alter in terms of protein structure and function. <b>Abbreviations</b>: ATG: autophagy-related; Cα: alpha carbon; CG: coarse-grained; CHARMM: Chemistry at Harvard macromolecular mechanics; CONAN: contact analysis; FUNDC1: FUN14 domain containing 1; FYCO1: FYVE and coiled-coil domain containing 1; GABARAP: GABA type A receptor-associated protein; GROMACS: Groningen machine for chemical simulations; HP: hydrophobic pocket; LIR: LC3 interacting region; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 B; MD: molecular dynamics; OPTN: optineurin; OSF: open software foundation; PE: phosphatidylethanolamine, PLEKHM1: pleckstrin homology domain-containing family M 1; PSN: protein structure network; PTM: post-translational modification; SA: structural alphabet; SLiM: short linear motif; SQSTM1/p62: sequestosome 1; WT: wild-type.