bims-apauto 2021-08-29 papers

Issue of 2021–08–29
seven papers selected by
Su Hyun Lee, Seoul National University

Autophagy. 2021 Aug 23. 1-19

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity.

Belinda S Hall, Scott J Dos Santos, Louise Tzung-Harn Hsieh, Maria Manifava, Marie-Thérèse Ruf, Gerd Pluschke, Nicholas Ktistakis, Rachel E Simmonds.

The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3-/-/perk-/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A1; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.

Keywords: Buruli ulcer; RB1CC1/FIP200; SQSTM1/p62; eif2s1/eIF2α; integrated stress response; mislocalized proteins; mycolactone; translocation inhibitor; ulk1

DOI: https://doi.org/10.1080/15548627.2021.1961067

PeerJ. 2021 ;9 e11968

Development and validation of a novel survival model for acute myeloid leukemia based on autophagy-related genes.

Li Huang, Lier Lin, Xiangjun Fu, Can Meng.

Background: Acute myeloid leukemia (AML) is one of the most common blood cancers, and is characterized by impaired hematopoietic function and bone marrow (BM) failure. Under normal circumstances, autophagy may suppress tumorigenesis, however under the stressful conditions of late stage tumor growth autophagy actually protects tumor cells, so inhibiting autophagy in these cases also inhibits tumor growth and promotes tumor cell death.
Methods: AML gene expression profile data and corresponding clinical data were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, from which prognostic-related genes were screened to construct a risk score model through LASSO and univariate and multivariate Cox analyses. Then the model was verified in the TCGA cohort and GEO cohorts. In addition, we also analyzed the relationship between autophagy genes and immune infiltrating cells and therapeutic drugs.
Results: We built a model containing 10 autophagy-related genes to predict the survival of AML patients by dividing them into high- or low-risk subgroups. The high-risk subgroup was prone to a poorer prognosis in both the training TCGA-LAML cohort and the validation GSE37642 cohort. Univariate and multivariate Cox analysis revealed that the risk score of the autophagy model can be used as an independent prognostic factor. The high-risk subgroup had not only higher fractions of CD4 naïve T cell, NK cell activated, and resting mast cells but also higher expression of immune checkpoint genes CTLA4 and CD274. Last, we screened drug sensitivity between high- and low-risk subgroups.
Conclusion: The risk score model based on 10 autophagy-related genes can serve as an effective prognostic predictor for AML patients and may guide for patient stratification for immunotherapies and drugs.

Keywords: Acute myeloid leukemia; Autophagy; GEO; Risk model; TCGA

DOI: https://doi.org/10.7717/peerj.11968

Autophagy. 2021 Aug 25. 1-2

Endolysosome and autophagy dysfunction in Alzheimer disease.

Christy Hung, Frederick J Livesey.

Abnormalities of the neuronal endolysosome and macroautophagy/autophagy system are an early and prominent feature of Alzheimer disease (AD). SORL1 is notable as a gene in which mutations are causal for a rare, autosomal dominant form of AD, and also variants that increase the risk of developing the common form of late-onset AD. In our recent study, we used patient-derived stem cells and CRISPR engineering to study the effects of SORL1 mutations on the endolysosome and autophagy system in human forebrain neurons. SORL1 mutations causal for monogenic AD are typically truncating mutations, and we found, using stem cells generated from an individual with dementia due to a heterozygous SORL1 truncation mutation, that this class of mutation results in SORL1 haploinsufficiency. Reducing SORL1 protein by half results in disrupted endosomal trafficking in patient-derived neurons, which we confirmed by studying the endolysosomal system in isogenic CRISPR-engineered SORL1 heterozygous null neurons. We also found that SORL1 homozygous null neurons develop more severe phenotypes, with endosome abnormalities, lysosome dysfunction and defects in the degradative phase of autophagy. Endolysosome and autophagy defects in SORL1 mutant neurons are dependent on APP, a key AD gene, as they are rescued by extracellular antisense oligonucleotides that reduce APP protein.

Keywords: Alzheimer’s disease; autophagy; endosome; live-cell imaging; lysosome

DOI: https://doi.org/10.1080/15548627.2021.1963630

Autophagy. 2021 Aug 25. 1-22

Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib.

Zhifei Xu, Ying Jin, Zizheng Gao, Yan Zeng, Jiangxia Du, Hao Yan, Xueqin Chen, Li Ping, Nengming Lin, Bo Yang, Qiaojun He, Peihua Luo.

Excessive macroautophagy/autophagy is one of the causes of cardiomyocyte death induced by cardiovascular diseases or cancer therapy, yet the underlying mechanism remains unknown. We and other groups previously reported that autophagy might contribute to cardiomyocyte death caused by sunitinib, a tumor angiogenesis inhibitor that is widely used in clinic, which may help to understand the mechanism of autophagy-induced cardiomyocyte death. Here, we found that sunitinib-induced autophagy leads to apoptosis of cardiomyocyte and cardiac dysfunction as the cardiomyocyte-specific Atg7-/+ heterozygous mice are resistant to sunitinib. Sunitinib-induced maladaptive autophagy selectively degrades the cardiomyocyte survival mediator CCN2 (cellular communication network factor 2) through the TOLLIP (toll interacting protein)-mediated endosome-related pathway and cardiomyocyte-specific knockdown of Ccn2 through adeno-associated virus serotype 9 (AAV9) mimics sunitinib-induced cardiac dysfunction in vivo, suggesting that the autophagic degradation of CCN2 is one of the causes of sunitinib-induced cardiotoxicity and death of cardiomyocytes. Remarkably, deletion of Hmgb1 (high mobility group box 1) inhibited sunitinib-induced cardiomyocyte autophagy and apoptosis, and the HMGB1-specific inhibitor glycyrrhizic acid (GA) significantly mitigated sunitinib-induced autophagy, cardiomyocyte death and cardiotoxicity. Our study reveals a novel target protein of autophagic degradation in the regulation of cardiomyocyte death and highlights the pharmacological inhibitor of HMGB1 as an attractive approach for improving the safety of sunitinib-based cancer therapy.

Keywords: Autophagy; CCN2 degradation; HMGB1; TOLLIP; cardiac dysfunction; glycyrrhizic acid; sunitinib

DOI: https://doi.org/10.1080/15548627.2021.1965712

Nat Commun. 2021 08 25. 12(1): 5058

Combined intermittent fasting and ERK inhibition enhance the anti-tumor effects of chemotherapy via the GSK3β-SIRT7 axis.

Xiaolong Tang, Guo Li, Lei Shi, Fengting Su, Minxian Qian, Zuojun Liu, Yuan Meng, Shimin Sun, Ji Li, Baohua Liu.

Dietary interventions such as intermittent fasting (IF) have emerged as an attractive strategy for cancer therapies; therefore, understanding the underlying molecular mechanisms is pivotal. Here, we find SIRT7 decline markedly attenuates the anti-tumor effect of IF. Mechanistically, AMP-activated protein kinase (AMPK) phosphorylating SIRT7 at T263 triggers further phosphorylation at T255/S259 by glycogen synthase kinase 3β (GSK3β), which stabilizes SIRT7 by decoupling E3 ligase UBR5. SIRT7 hyperphosphorylation achieves anti-tumor activity by disrupting the SKP2-SCF E3 ligase, thus preventing SKP2-mediated K63-linked AKT polyubiquitination and subsequent activation. In contrast, GSK3β-SIRT7 axis is inhibited by EGF/ERK2 signaling, with ERK2 inactivating GSK3β, thus accelerating SIRT7 degradation. Unfavorably, glucose deprivation or chemotherapy hijacks the GSK3β-SIRT7 axis via ERK2, thus activating AKT and ensuring survival. Notably, Trametinib, an FDA-approved MEK inhibitor, enhances the efficacy of combination therapy with doxorubicin and IF. Overall, we have revealed the GSK3β-SIRT7 axis that must be fine-tuned in the face of the energetic and oncogenic stresses in malignancy.

DOI: https://doi.org/10.1038/s41467-021-25274-3

Autophagy. 2021 Aug 23. 1-3

LC3B phosphorylation: autophagosome's ticket for a ride toward the cell nucleus.

Jose L Nieto-Torres, Sandra E Encalada, Malene Hansen.

Macroautophagy/autophagy is a multi-step process that leads to cargo degradation via the fusion of hydrolases-containing lysosomes with cargo-loaded autophagosomes. For this process to occur, autophagosomes are directionally transported by molecular motors toward the nucleus, where they fuse with lysosomes for cargo degradation. The molecular basis for this regulation, including the cell machinery required for this directional transport, has not been fully identified. Using a combination of proteomic and live-imaging approaches in mammalian cells, including primary neurons, we describe that the phosphorylation of the autophagosome protein Atg8/LC3B by the Hippo kinase STK4/MST1, an event we previously reported to be required for autophagy completion, reduces the binding of the transport-related protein FYCO1 to MAP1LC3B/LC3B. This event in turn allows the proficient microtubule-based transport of autophagosomes toward the perinuclear area, thus facilitating the contact of autophagosomes with lysosomes. In the absence of LC3B phosphorylation, autophagosomes undergo aberrant transport including increased movement toward the cell periphery resulting in reduced autophagosome-lysosome colocalization. Thus, LC3B phosphorylation modulates the directional transport of autophagosomes to meet with lysosomes in the perinuclear area, a crucial event in ensuring autophagic degradation of cargo.

Keywords: Atg8; FYCO1; LC3B; STK4; MST1; autophagosome transport; phosphorylation

DOI: https://doi.org/10.1080/15548627.2021.1961073

Science. 2021 Jul 23. 373(6553): 413-419

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

DOI: https://doi.org/10.1126/science.abe7565