Abstract:
:Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.
journal_name
Autophagyjournal_title
Autophagyauthors
Mercer CA,Kaliappan A,Dennis PBdoi
10.4161/auto.5275subject
Has Abstractpub_date
2008-02-01 00:00:00pages
185-94issue
2eissn
1554-8627issn
1554-8635pii
5275journal_volume
4pub_type
杂志文章相关文献
Autophagy文献大全abstract::SNCA/α-synuclein aggregation plays a crucial role in synucleinopathies such as Parkinson disease and dementia with Lewy bodies. Aggregating and nonaggregating SNCA species are degraded by the autophagy-lysosomal pathway (ALP). Previously, we have shown that the ALP is not only responsible for SNCA degradation but is a...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.36436
更新日期:2014-01-01 00:00:00
abstract::Ferroptosis is a form of regulated cell death caused by iron accumulation and oxidative injury. BECN1 is a key regulator of macroautophagy/autophagy, a catabolic process of degradation induced by starvation or other stressors. Our recent findings reveal a novel alternative mechanism by which BECN1 can promote ferropto...
journal_title:Autophagy
pub_type: 评论,杂志文章
doi:10.1080/15548627.2018.1513758
更新日期:2018-01-01 00:00:00
abstract::SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we repor...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2017.1291479
更新日期:2017-05-04 00:00:00
abstract::Cutaneous inflammation from UV radiation exposure causes epidermal damage, cellular infiltration, and secretion of pro-inflammatory mediators that exacerbate tissue destruction. Recovery is mediated chiefly by anti-inflammatory M2 macrophages that suppress inflammation and augment epidermal regeneration. Vitamin D ena...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2019.1569298
更新日期:2019-05-01 00:00:00
abstract::To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesi...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.26503
更新日期:2013-11-01 00:00:00
abstract::In macroautophagy (hereafter autophagy), a morphological hallmark is the formation of double-membrane vesicles called autophagosomes that sequester and deliver cytoplasmic components to the lysosome/vacuole for degradation. This process begins with an initial sequestering compartment, the phagophore, which expands int...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.6.7.13009
更新日期:2010-10-01 00:00:00
abstract::Autophagy plays critical roles in plant responses to stress. In contrast to the wealth of information concerning the core process of plant autophagosome assembly, our understanding of the regulation of autophagy is limited. In this study, we demonstrated that transcription factor HsfA1a played a critical role in tomat...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2015.1098798
更新日期:2015-11-02 00:00:00
abstract::Bulk degradation and nutrient recycling are events associated with autophagy. The core components of the autophagy machinery have been elucidated recently using molecular and genetic approaches. In particular, two ubiquitin-like proteins, ATG8 and ATG12, which conjugate with phosphatidylethanolamine (PE) and ATG5, res...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1105/tpc.110.075333
更新日期:2010-08-01 00:00:00
abstract::Pathological developments leading to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are associated with misbehavior of several key proteins, such as SOD1 (superoxide dismutase 1), TARDBP/TDP-43, FUS, C9orf72, and dipeptide repeat proteins generated as a result of the translation of th...
journal_title:Autophagy
pub_type: 杂志文章,评审
doi:10.1080/15548627.2017.1384889
更新日期:2017-01-01 00:00:00
abstract::LC3 is a widely used marker of autophagosomes in mammalian cells. However, in addition to its autophagosomal localization, GFP-LC3 is often found associated with protein aggregates that are formed in an autophagy-independent manner. In addition, LC3 directly interacts with p62/SQSTM1 (hereafter named p62), a common co...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.6823
更新日期:2008-11-01 00:00:00
abstract::The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, ...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.6.8.13429
更新日期:2010-11-01 00:00:00
abstract::The critical role of the cellular autophagy pathway in viral infection and pathogenesis has become increasingly apparent. Mounting evidences suggest that viruses have developed different strategies to meticulously modulate intracellular autophagy for their own benefits, thereby either promoting efficient viral replica...
journal_title:Autophagy
pub_type: 杂志文章,评审
doi:10.4161/auto.5210
更新日期:2008-04-01 00:00:00
abstract::Under stress conditions cells activate different response pathways which result in cell survival or apoptosis depending on: (1) the nature of the insults, (2) the type, if acute or chronic stress, and (3) how long the stress persists. Generally, autophagy is induced early to sustain cell survival and inhibit cell deat...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.20671
更新日期:2012-08-01 00:00:00
abstract::Mitochondrial dysfunction is associated with the occurrence of a variety of neurodegenerative diseases, especially Alzheimer disease (AD). As a mitochondrial quality control process, mitophagy is greatly inhibited in AD; increasing evidence shows that the induction of mitophagy is an effective therapeutic intervention...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2020.1860542
更新日期:2020-12-20 00:00:00
abstract::DBI/ACBP (diazepam binding protein, acyl-CoA binding protein) participates in the regulation of fatty acid metabolism when it is localized within cells, whereas outside of cells it acts as a diazepam-binding protein. Recent results indicate that many different mammalian cell types release DBI/ACBP upon in vitro or in ...
journal_title:Autophagy
pub_type: 评论,杂志文章
doi:10.1080/15548627.2019.1662585
更新日期:2019-11-01 00:00:00
abstract::Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan ...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2019.1596483
更新日期:2019-11-01 00:00:00
abstract::Autophagy and apoptosis are 2 fundamental biological mechanisms that may cooperate or be antagonistic, although both are involved in deciding the fate of cells in physiological or pathological conditions. These 2 mechanisms coexist simultaneously in cells and share common upstream signals and stimuli. Autophagy and ap...
journal_title:Autophagy
pub_type: 杂志文章,评审
doi:10.1080/15548627.2016.1256522
更新日期:2017-03-04 00:00:00
abstract::The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this stud...
journal_title:Autophagy
pub_type: 评论,社论
doi:10.1080/15548627.2016.1173802
更新日期:2016-07-02 00:00:00
abstract::Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesic...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.4129
更新日期:2007-09-01 00:00:00
abstract::Helicobacter pylori is a gram negative pathogen that infects at least half of the world's population and is associated not only with gastric cancer but also with other diseases such as gastritis and peptic ulcers. Indeed, H. pylori is considered the single most important risk factor for the development of gastric canc...
journal_title:Autophagy
pub_type: 杂志文章,评审
doi:10.4161/auto.6.1.10222
更新日期:2010-01-01 00:00:00
abstract::Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulator...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2017.1389357
更新日期:2018-01-01 00:00:00
abstract::Mesenchymal stem cell (MSC) transplantation has been widely applied as a potential therapeutic for multiple diseases. However, the underlying therapeutic mechanisms are not fully understood, especially the paradox between the low survival rate of transplanted cells and the beneficial therapeutic effects generated by t...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.1080/15548627.2020.1717128
更新日期:2020-12-01 00:00:00
abstract::Significant progress has been made over recent years in defining the normal progression and regulation of autophagy, particularly in cultured mammalian cells and yeast model systems. However, apart from a few notable exceptions, our understanding of the physiological roles of autophagy has lagged behind these advances...
journal_title:Autophagy
pub_type: 杂志文章,评审
doi:10.4161/auto.5782
更新日期:2008-07-01 00:00:00
abstract::Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline ...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.7.2.14043
更新日期:2011-02-01 00:00:00
abstract::The understanding of the membrane flow process during autophagosome formation is essential to illuminate the role of autophagy under various disease-causing conditions. Atg9 is the only identified integral membrane protein required for autophagosome formation, and it is thought to cycle between the membrane sources an...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.5.3.7699
更新日期:2009-04-01 00:00:00
abstract::Autophagy is a highly conserved degradation pathway for intracellular macromolecules and organelles. Among those characterized autophagy regulators, the ubiquitin-like protein Atg8 is found to be a membrane modifier that both regulates biogenesis of transport vesicles and interacts with the cargo receptor Atg19 for se...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.5.4.7696
更新日期:2009-05-01 00:00:00
abstract::Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 ligh...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.6.8.13427
更新日期:2010-11-01 00:00:00
abstract::This study summarizes our most recent findings on the mechanisms underlying the cadmium-induced death of mesangial cells, which leads to nephrotoxicity. Multiple pathways participate in cadmium-induced nephrotoxicity. In the ROS-GSK-3beta autophagy pathway, cadmium induces ROS most likely from the mitochondria, and th...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.5.4.8311
更新日期:2009-05-01 00:00:00
abstract::Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human beta-amyloid peptide ...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.4776
更新日期:2007-11-01 00:00:00
abstract::Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradati...
journal_title:Autophagy
pub_type: 杂志文章
doi:10.4161/auto.6657
更新日期:2008-10-01 00:00:00