Protein Synthesis and Degradation
A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy
Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells, but these studies can be challenging because autophagosomes disintegrate easily. In the yeast Saccharomyces cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation.α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α
α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor–differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α.Proteaphagy is specifically regulated and requires factors dispensable for general autophagy
Changing physiological conditions can increase the need for protein degradative capacity in eukaryotic cells. Both the ubiquitin-proteasome system and autophagy contribute to protein degradation. However, these processes can be differently regulated depending on the physiological conditions. Strikingly, proteasomes themselves can be a substrate for autophagy. The signals and molecular mechanisms that govern proteasome autophagy (proteaphagy) are only partly understood. Here, we used immunoblots, native gel analyses, and fluorescent microscopy to understand the regulation of proteaphagy in response to genetic and small molecule-induced perturbations.Development of a specific live-cell assay for native autophagic flux
Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling.Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site
Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme.Use of the LC3B-fusion technique for biochemical and structural studies of proteins involved in the N-degron pathway
The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification.The RING-type E3 ligase RNF186 ubiquitinates Sestrin-2 and thereby controls nutrient sensing
Nutrient sensing is a critical cellular process controlling metabolism and signaling. mTOR complex 1 (mTORC1) is the primary signaling hub for nutrient sensing and, when activated, stimulates anabolic processes while decreasing autophagic flux. mTORC1 receives nutrient status signals from intracellular amino acid sensors. One of these sensors, Sestrin-2, functions as an intracellular sensor of cytosolic leucine and inhibitor of mTORC1 activity. Genetic studies of Sestrin-2 have confirmed its critical role in regulating mTORC1 activity, especially in the case of leucine starvation.Deficiency of the autophagy gene ATG16L1 induces insulin resistance through KLHL9/KLHL13/CUL3-mediated IRS1 degradation
Connections between deficient autophagy and insulin resistance have emerged, however, the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO mouse embryonic fibroblasts (MEFs)), an essential autophagy gene, and observed deficient insulin and insulin-like growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of insulin receptor substrate-1 (IRS1), pivotal to insulin signaling, whereas IRS1myc overexpression recovered downstream insulin signaling.Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins
Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids.Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy
The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF).Introduction to the Thematic Minireview Series: Autophagy
Autophagy is a highly conserved, tightly regulated cellular process that degrades intracellular constituents via lysosomes. Autophagy mediates many normal cellular functions and is dysregulated in numerous diseases. This Thematic Series consists of five Minireviews that highlight selected topics of current autophagy research ranging from the molecular mechanisms and regulation of autophagy to the roles of autophagy in health and disease.The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43
Connexin-43 (Cx43, also known as GJA1) is the most ubiquitously expressed connexin isoform in mammalian tissues. It forms intercellular gap junction (GJ) channels, enabling adjacent cells to communicate both electrically and metabolically. Cx43 is a short-lived protein which can be quickly degraded by the ubiquitin-dependent proteasomal, endolysosomal, and autophagosomal pathways. Here, we report that the ubiquitin-specific peptidase 8 (USP8) interacts with and deubiquitinates Cx43. USP8 reduces both multiple monoubiquitination and polyubiquitination of Cx43 to prevent autophagy-mediated degradation.Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4
Turnover of the 26S proteasome by autophagy is an evolutionarily conserved process that governs cellular proteolytic capacity and eliminates inactive particles. In most organisms, proteasomes are located in both the nucleus and cytoplasm. However, the specific autophagy routes for nuclear and cytoplasmic proteasomes are unclear. Here, we investigate the spatial control of autophagic proteasome turnover in budding yeast (Saccharomyces cerevisiae). We found that nitrogen starvation–induced proteasome autophagy is independent of known nucleophagy pathways but is compromised when nuclear protein export is blocked.Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells
In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis.Function of inhibitor of Bruton's tyrosine kinase isoform α (IBTKα) in nonalcoholic steatohepatitis links autophagy and the unfolded protein response
Nonalcoholic fatty liver disease (steatosis) is the most prevalent liver disease in the Western world. One of the advanced pathologies is nonalcoholic steatohepatitis (NASH), which is associated with induction of the unfolded protein response (UPR) and disruption of autophagic flux. However, the mechanisms by which these processes contribute to the pathogenesis of human diseases are unclear. Herein, we identify the α isoform of the inhibitor of Bruton's tyrosine kinase (IBTKα) as a member of the UPR, whose expression is preferentially translated during endoplasmic reticulum (ER) stress.Zinc starvation induces autophagy in yeast
Zinc is an essential nutrient for all forms of life. Within cells, most zinc is bound to protein. Because zinc serves as a catalytic or structural cofactor for many proteins, cells must maintain zinc homeostasis under severely zinc-deficient conditions. In yeast, the transcription factor Zap1 controls the expression of genes required for uptake and mobilization of zinc, but to date the fate of existing zinc-binding proteins under zinc starvation remains poorly understood. Autophagy is an evolutionarily conserved cellular degradation/recycling process in which cytoplasmic proteins and organelles are sequestered for degradation in the vacuole/lysosome.Recycling of iron via autophagy is critical for the transition from glycolytic to respiratory growth
Autophagy is a bulk degradation process conserved from yeast to mammals. To examine the roles of autophagy in cellular metabolism, we generated autophagy-defective (atg) mutants in the X2180-1B strain background. We compared the growth of wild-type (WT) and atg cells in minimal (synthetic dextrose, SD) and rich (yeast extract/peptone/dextrose, YEPD) medium, and we found that mutations in the core autophagy machinery result in defects in the diauxic shift, the transition from fermentation to respiratory growth upon glucose depletion, specifically in SD.Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (Cdc37) and Thereby Disrupts the Stability of Cdc37 Client Proteins
The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate.Sequestosome 1/p62 Protein Is Associated with Autophagic Removal of Excess Hepatic Endoplasmic Reticulum in Mice
Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not.Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles
The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol.Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response
Background: Misfolded proteins are a ubiquitous and clinically relevant threat to cells.Results: Arsenite stress in yeast leads to increased protein degradation and reduced protein production.Conclusion: Reduction in ribosome abundance is a novel, rapid, effective, and reversible stress response against misfolded proteins.Significance: These results provide the basis for further characterization of a potentially important stress response pathway.Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy
Background: Several rare lung proteinopathies are characterized by lung fibrosis due to accumulation of misfolded proteins in epithelial cells.Results: The PiZ mouse models the pathological characteristics of these lung proteinopathies, and the pathology is ameliorated by autophagy enhancing therapies.Conclusion: Autophagy represents a key proteostasis mechanism for lung proteinopathies and a potential therapeutic target.Significance: The PiZ mouse is an attractive animal model of lung proteinopathies.The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy
Background: Autophagosome formation is mediated by multiple autophagy-related (Atg) proteins.Results: Essential Atg proteins of K. marxianus, which have superior thermostability and solubility, are identified.Conclusion: K. marxianus can be used as a novel organism to study autophagy.Significance: K. marxianus proteins are broadly applicable as tools for in vitro studies, not only in autophagy field but also in other fields.Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi
Background: Successful colonization of humans by the parasite Trypanosoma cruzi critically depends on intact parasite autophagy and autophagin-2.Results: T. cruzi autophagin-2, in contrast to human autophagin-1, exhibits substantial preference toward the unnatural amino acid cyclohexylalanine in the P2 position.Conclusion: Selective fluorogenic substrates, which are only recognized by T. cruzi autophagin-2, were synthesized.Significance: These selective substrates have a potential for screening of T. cruzi autophagin-2 specific inhibitors.Potassium Bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)) Induces Apoptosis and Pyroptosis and Disrupts the P62-HDAC6 Protein Interaction to Suppress the Acetylated Microtubule-dependent Degradation of Autophagosomes
Background: BpV(phen) is an insulin-mimetic small molecule.Results: We demonstrate that bpV(phen) reduces the stability of p62 in a proteasome-dependent way to activate HDAC6 to inhibit autophagy and induce apoptosis and pyroptosis.Conclusion: We propose that bpV(phen) inhibits autophagy through p62.Significance: We provide insights into a novel function of bpV(phen) and p62 in autophagy.
