Multiple Genes Core to ERAD, Macroautophagy and Lysosomal Degradation Pathways Participate in the Proteostasis Response in α1-Antitrypsin Deficiency.

BACKGROUND & AIMS: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD. METHODS: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems. RESULTS: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria. CONCLUSIONS: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.

Molecular targets and approaches to restore autophagy and lysosomal capacity in neurodegenerative disorders.

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity.

TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41.

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

Fluopack screening platform for unbiased cellular phenotype profiling.

Gene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization.

TMEM41B is a novel regulator of autophagy and lipid mobilization.

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and ß-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9.

Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies.

Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

Revisiting autophagy addiction of tumor cells.

Inhibition of autophagy has been widely explored as a potential therapeutic intervention for cancer. Different factors such as tumor origin, tumor stage and genetic background can define a tumor's response to autophagy modulation. Notably, tumors with oncogenic mutations in KRAS were reported to depend on macroautophagy in order to cope with oncogene-induced metabolic stress. Our recent report details the unexpected finding that autophagy is dispensable for KRAS-driven tumor growth in vitro and in vivo. Additionally, we clarify that the antitumorigenic effects of chloroquine, a frequently used nonspecific inhibitor of autophagy, are not connected to the inhibition of macroautophagy. Our data suggest that caution should be exercised when using chloroquine and its analogs to decipher the roles of autophagy in cancer.

Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy.

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.

RAD001 enhances the potency of BEZ235 to inhibit mTOR signaling and tumor growth.

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.

Identification of elongation factor G as the conserved cellular target of argyrin B.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

Quantitative visualization of autophagy induction by mTOR inhibitors.

Autophagy is a catabolic pathway that degrades bulk cytosol in lysosomal compartments enabling amino acids and fatty acids to be recycled. One of the key regulators of autophagy is the mammalian target of rapamycin (mTOR), a conserved serine/threonine kinase which suppresses the initiation of the autophagic process when nutrients, growth factors, and energy are available. Inhibition of mTOR, e.g., by small molecules such as rapamycin, results in activation of autophagy. To quantify autophagy induction by mTOR inhibitors, we use an mCherry-GFP-LC3 reporter which is amenable to retroviral delivery into mammalian cells, stable expression, and analysis by fluorescence microscopy. Here, we describe our imaging protocol and image recognition algorithm to visualize and measure changes in the autophagic pathway.

Relieving autophagy and 4EBP1 from rapamycin resistance.

The mammalian target of rapamycin complex 1 (mTORC1) is a multiprotein signaling complex regulated by oncogenes and tumor suppressors. Outputs downstream of mTORC1 include ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E (eIF4E), and autophagy, and their modulation leads to changes in cell growth, proliferation, and metabolism. Rapamycin, an allosteric mTORC1 inhibitor, does not antagonize equally these outputs, but the reason for this is unknown. Here, we show that the ability of rapamycin to activate autophagy in different cell lines correlates with mTORC1 stability. Rapamycin exposure destabilizes mTORC1, but in cell lines where autophagy is drug insensitive, higher levels of mTOR-bound raptor are detected than in cells where rapamycin stimulates autophagy. Using small interfering RNA (siRNA), we find that knockdown of raptor relieves autophagy and the eIF4E effector pathway from rapamycin resistance. Importantly, nonefficacious concentrations of an ATP-competitive mTOR inhibitor can be combined with rapamycin to synergistically inhibit mTORC1 and activate autophagy but leave mTORC2 signaling intact. These data suggest that partial inhibition of mTORC1 by rapamycin can be overcome using combination strategies and offer a therapeutic avenue to achieve complete and selective inhibition of mTORC1.

Role of the lectin VIP36 in post-ER quality control of human alpha1-antitrypsin.

The leguminous-type (L-type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high-mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar-binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein alpha1-antitrypsin (alpha1-AT) as a cargo of VIP36. The VIP36/alpha1-AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high-mannose form of alpha1-AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of alpha1-AT were inactivated by mutagenesis. Silencing VIP36 accelerated alpha1-AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and alpha1-AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post-ER quality control of alpha1-AT.

Bidirectional transport of amino acids regulates mTOR and autophagy.

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin.

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

Deletion of 3 residues from the C-terminus of MCFD2 affects binding to ERGIC-53 and causes combined factor V and factor VIII deficiency.

Combined factor V and factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder. F5F8D is genetically linked to mutations in the transmembrane lectin ERGIC-53 and its soluble interaction partner MCFD2. The ERGIC-53/MCFD2 protein complex functions as transport receptor of coagulation factors V and VIII by mediating their export from the endoplasmic reticulum (ER). Here, we studied a F5F8D patient who was found to be a compound heterozygote for 2 novel mutations in MCFD2: a large deletion of 8.4 kb eliminating the 5'UTR of the gene and a nonsense mutation resulting in the deletion of only 3 amino acids (DeltaSLQ) from the C-terminus of MCFD2. Biochemical and structural analysis of the DeltaSLQ mutant demonstrated impaired binding to ERGIC-53 due to modification of the 3-dimensional structure of MCFD2. Our results highlight the importance of the ERGIC-53/MCFD2 protein interaction for the efficient secretion of coagulation factors V and VIII.

Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL, and VIP36.

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.