TMEM74, a lysosome and autophagosome protein, regulates autophagy
Section snippets
Materials and methods
Materials. Earle’s balanced salt solution (EBSS), bafilomycin A1, monodansylcadaverine (MDC), and a monoclonal antibody against β-actin were obtained from Sigma (USA). cDNA libraries of cancer tissues were obtained from Shanghai Genomics, Inc. Antibodies against cathepsinD and MAP-LC3 were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc.). LysoTracker Red was obtained from Molecular Probes (USA). ER-DsRed plasmid was obtained from Clontech (USA). Alexa Fluor 780-labeled IgG secondary
Cloning, bioinformatics analysis and expression profile of human TMEM74
The human TMEM74 cDNA clone [15] was directly isolated from a human esophagus cDNA library and found to be 2087 base pairs long with an in-frame stop codon upstream of the putative ATG start codon. The sequence surrounding the ATG start codon largely followed the Kozak consensus rule. The open reading frame encoded 305 amino acids with a predicted molecular mass of 33.33 kDa and an isoelectric point of 4.87. The full-length cDNA and predicted amino acid sequence of TMEM74 are shown in Fig. 1A.
Discussion
Our data suggest that TMEM74 induces autophagic characters of cells, including cytoplasmic vacuolization under TEM (Fig. 2A), punctate distribution of GFP-LC3 (Fig. 2B) and MDC staining (Fig. 2D), and conversion of LC3-I to LC3-II (Fig. 2E). The autophagic response requires delivery of the contents sequestered by the autophagosome to the lysosome, whereupon degradation occurs [18]. Cytoplasmic vacuolization might reflect an increase in autophagic activity or a build-up of nonfused
Acknowledgments
Contract grant sponsor: China High Tech 863 Program; Contract Grant No. 2006AA02A305.
References (23)
- et al.
Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy
Eur. J. Cell Biol.
(2000) Autophagy: a regulated bulk degradation process inside cells
Biochem. Biophys. Res. Commun.
(2004)Autophagy and related mechanisms of lysosome-mediated protein degradation
Trends Cell Biol.
(1994)Methods for monitoring autophagy
Int. J. Biochem. Cell Biol.
(2004)- et al.
Overexpression of the novel human gene, nuclear apoptosis-inducing factor 1, induces apoptosis
Int. J. Biochem. Cell Biol.
(2006) - et al.
Identification of subcellular compartments involved in biosynthetic processing of cathepsin D
J. Biol. Chem.
(1992) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense
Cell
(2005)- et al.
Autophagy in the eukaryotic cell
Eukaryot. Cell
(2002) - et al.
Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after glucagon administration
Am. J. Pathol.
(1968) - et al.
Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization
J. Cell Biol.
(1994)
Autophagy as a regulated pathway of cellular degradation
Science
Cited by (48)
RNA sequence analysis of nasopharyngeal swabs from asymptomatic and mildly symptomatic patients with COVID-19
2022, International Journal of Infectious DiseasesCitation Excerpt :We found an upregulation of MAPILC3C in the symptomatic group (Fig. 6). Those genes are positive regulators in the autophagosome formation process (Bonam et al., 2020; Yu et al., 2008). The MAP1LC3C protein is a member of the MAP1LC3 protein family, which plays part in the autophagosome initiation (Bonam et al., 2020).
Mycophenolic acid (MPA) modulates host cellular autophagy progression in sub genomic dengue virus-2 replicon cells
2019, Microbial PathogenesisCitation Excerpt :The upregulated ULK2 gene is crucial for the autophagy initiation [23] and Cathepsin D (CTSD) is an important autophagy marker [5] in DENV replication. Inhibition of CTSD impairs the autophagy flux and TMEM74 protein over-expression has a role in the autophagy cellular process [24]. Also, TMEM 74 is involved in the initial stage of functional autophagosome formation [24].
Autophagy regulatory molecule, TMEM74, interacts with BIK and inhibits BIK-induced apoptosis
2017, Cellular SignallingCitation Excerpt :TMEM74, as a novel lysosome transmembrane protein, was firstly identified in our lab, TMEM74 is conserved in humans, M. mulatta, B. taurus, R. norvegicus, and M. musculus, however, no obvious homology with any known genes or proteins has not been found or confirmed so far. Our lab firstly performed the functional report about TMEM74 and demonstrated its autophagy induction effect [14,15], whereas the deeper mechanisms about the TMEM74 function still remain unclear. Bik, containing only one of the signature domains of the Bcl-2 family, the BH3 domain, mediates the induction of cell apoptosis by Bax-dependent mitochondria pathway.
Autophagic cell death of human hepatoma G2 cells mediated by procyanidins from Castanea mollissima Bl. Shell-induced reactive oxygen species generation
2014, Chemico-Biological InteractionsCitation Excerpt :Autophagy orchestrates cells to self-digest their own long-lived proteins, organelles or DNA, underscoring its critical role in cellular homeostasis [9]. However, a poor understanding about the molecular mechanisms of autophagy and vesicle trafficking, especially the biogenesis and turnover of autophagosomes remains [10]. Previous reports have indicated that autophagy-related gene (Atg) products act as the core machinery in the initiation, processing, and maturation of autophagy [11].
The Therapeutic Effects of Dihydroartemisinin on Cisplatin-Resistant Gastric Cancer Cells
2022, Current Pharmaceutical Biotechnology