Development of a novel method for quantification of autophagic protein degradation by AHA labeling

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.     

Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters

The analysis of autophagy in cells and tissue has principally been performed via qualitative measures. These assays identify autophagosomes or measure the conversion of LC3I to LC3II. However, qualitative assays fail to quantitate the degradation of an autophagic substrate and therefore only indirectly measure an intact autophagic system. “Autophagic flux” can be measured using long-lived proteins that are degraded via autophagy. We developed a quantifiable luciferase reporter assay that measures the degradation of a long-lived polyglutamine protein aggregate, polyQ80-luciferase. Using this reporter, the induction of autophagy via starvation or rapamycin in cells preferentially decreases polyQ80-luciferase when compared with a nonaggregating polyQ19-luciferase after four hours of treatment. This response was both time- and concentration-dependent, prevented by autophagy inhibitors and absent in ATG5 knockout cells. We adapted this assay to living animals by electroporating polyQ19-luciferase and polyQ80-luciferase expression constructs into the right and left tibialis anterior (TA) muscles of mice, respectively. The change in the ratio of polyQ80-luciferase to polyQ19-luciferase signal before and after autophagic stimulation or inhibition was quantified via in vivo bioluminescent imaging. Following two days of starvation or treatment with intraperitoneal rapamycin, there was a ~35% reduction in the ratio of polyQ80:polyQ19-luciferase activity, consistent with the selective autophagic degradation of polyQ80 protein. This autophagic response in skeletal muscle in vivo was abrogated by co-treatment with chloroquine and in ATG16L1 hypomorphic mice. Our study demonstrates a method to quantify the autophagic flux of an expanded polyglutamine via luciferase reporters in vitro and in vivo.     

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ^TM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.     

Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells

Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP⁺RFP⁺ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.     

Selective removal of dishevelled by autophagy: A role of p62

Autophagy has long been viewed as a process to remove long-lived or misfolded protein aggregates and aging and damaged organelles. Our study identified a previously unknown function of autophagy in suppression of Wnt signaling. A signaling protein, Dishevelled (Dvl), can be degraded via the autophagy pathway upon starvation. In this selective degradation, p62/sequestosome-1 binds to ubiquitinated Dvl proteins and promotes Dvl aggregation. Intriguingly, LC3 can also directly interact with Dvl. The studies on the mechanism for autophagic clearance of Dishevelled led to several interesting findings.     

Proteomics analysis of starved cells revealed Annexin A1 as an important regulator of autophagic degradation

Macroautophagy is involved in the bulk degradation of long-lived cytosolic proteins and subcellular organelles, which is important for the survival of cells during starvation. To identify potential players of the autophagy process, we subjected HCT116 cells cultured in complete medium and in Earle’s balanced salt solution to proteomics analysis. In approximately 1500 protein spots detected, we characterized 52 unique proteins, whose expression levels were significantly changed following starvation. Notably, we found that Annexin A1 was significantly upregulated following starvation at both mRNA and protein levels. Inhibition of Annexin A1 expression with specific siRNA did not alter starvation-induced autophagy as measured by the level of lipidated LC3, but significantly reversed autophagy degradation as measured by the level of p62/SQSTM 1. Thus Annexin A1 seemed to be positively upregulated during starvation to promote autophagic degradation. Overall, the data presented in this study established a expression profile of the proteome in starved cells, which allowed the identification of proteins with potential significance in starvation-induced autophagy.     

NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.     

A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.     

Endogenous HMGB1 regulates autophagy

Autophagy clears long-lived proteins and dysfunctional organelles and generates substrates for adenosine triphosphate production during periods of starvation and other types of cellular stress. Here we show that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is a critical regulator of autophagy. Stimuli that enhance reactive oxygen species promote cytosolic translocation of HMGB1 and thereby enhance autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2. Mutation of cysteine 106 (C106), but not the vicinal C23 and C45, of HMGB1 promotes cytosolic localization and sustained autophagy. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin1 and sustaining autophagy. Thus, endogenous HMGB1 is a critical pro-autophagic protein that enhances cell survival and limits programmed apoptotic cell death.     

Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.     

A simple estimation of peroxisomal degradation with green fluorescent protein - an application for cell cycle analysis.

When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO-K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient-starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3-methyladenine suppressed the decay of GFP fluorescence (13-60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.     

Alfy-dependent elimination of aggregated proteins by macroautophagy: Can there be too much of a good thing?

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to nonselective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.Key words: autophagy, protein aggregates, neurodegeneration, Alfy, aggregation, degradation     

AMPKα promotes basal autophagy induction in Dictyostelium discoideum

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5′-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα⁻ cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα⁻ cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα⁻ cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK–ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, β-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα⁻ cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.     

Amino Acid Deprivation Induces Both Apoptosis and Autophagy in Murine C2C12 Muscle Cells

Apoptosis and autophagy are closely interconnected types of programmed cell death. In the present study, mouse C2C12 muscle cells were starved in Earle’s Balanced Salt Solution or treated with TNF-α and cycloheximide to induce autophagy and apoptosis, respectively. The majority of starved C2C12 cells underwent autophagy, as shown by LC3 processing, formation of autophagic vesicles and bulk degradation of long-lived proteins. However, some cells showed features of apoptosis including caspase-3 cleavage, chromatin condensation, DNA fragmentation and annexin V labeling. Caspase-3 cleavage was also induced in culture medium without serum, suggesting that serum withdrawal rather than amino acid deprivation triggered apoptosis. Starvation eliminated multiple pro-apoptotic proteins, but upregulated caspase-8, and rendered starved C2C12 cells much more susceptible to TNF-α/cycloheximide-induced apoptosis than non-starved cells. Our data suggest that amino acid deprivation of C2C12 cells induces a complex form of cell death with hallmarks of both apoptosis and autophagy.     

Alfy-dependent elimination of aggregated proteins by macroautophagy

Degradation of different cargo by macroautophagy is emerging as a highly selective process which relies upon specific autophagy receptors and adapter molecules that link the cargo with the autophagic molecular machinery. We have recently reported that the large phsophatidylinositol-3-phosphate (PtdIns(3)P)-binding protein Alfy (Autophagy-linked FYVE protein) is required for selective degradation of aggregated proteins. Although depletion of Alfy inhibits Atg5-dependent aggregate degradation, overexpression of Alfy results in Atg5-dependent aggregate clearance and neuroprotection. Alfy-mediated degradation requires the ability of Alfy to directly interact with Atg5. This ability to interact with the core autophagic machinery may cause Alfy to diminish the responsiveness to non-selective autophagic degradation as measured by long-lived protein degradation. Thus, increasing Alfy-mediated protein degradation may be beneficial in some organs, but may be detrimental in others.     

Glutaminolysis yields a metabolic by-product that stimulates autophagy

Autophagy is an intracellular degradative pathway that plays key roles in the homeostatic turnover of long-lived or damaged proteins and organelles, and in the survival of cells during starvation or other stressful conditions. We have uncovered an unexpected link between glutamine (Gln) metabolism and the regulation of autophagy. Our findings indicate that ammonia, generated from Gln deamination in mitochondria, functions as an autocrine- and/or paracrine-acting stimulator of autophagic flux.