Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

To investigate the ability of SAHA-induced TRAIL DR5-CTSB crosstalk to initiate the breast cancer autophagy, RTCA assay was performed to assess the effect of SAHA on breast cancer cells, and western blot and ELISA were used to verify the inductive effects on expression of CTSB. Breast cancer cells were transfected with TRAIL DR5 siRNA to block the function of TRAIL DR5. Cell viability and apoptosis of breast cancer cells were analyzed using a muse cell analyzer. The distribution of LC3-II in TRAIL DR5-silenced breast cancer cells treated with SAHA was observed by immunofluorescence microscopy, the mRNA levels of autophagy-related genes were detected by RNA microarray, and the activity of autophagy-related signaling pathways was screened by MAPK antibody array. Results indicated that SAHA did indeed repress the growth of breast cancer cell lines with inducing CTSB expression. Western blot and ELISA results indicated that TRAIL DR5 was involved in the expression of CTSB in SAHA-induced breast cancer cells. Cell viability and apoptosis assays showed that the inactivation of TRAIL DR5 can significantly inhibit the effects of SAHA. An immunofluorescence assay indicated that, with SAHA treatment, MDA-MB-231 and MCF-7 cells underwent apparent morphological changes. While SAHA was added in the TRAIL-DR5 blocked cells, the distribution of LC3-II signal was dispersed, the intensity of fluorescence signal was weaker than that of SAHA alone. RNA array indicated that SAHA significantly increased mRNA expression of autophagy marker LC3A/B whereas the change was significantly reversed in TRAIL DR5-silenced cells. The results of MAPK antibody array showed that SAHA and TRAIL DR5 could affect the activity of AKT1, AKT2, and TOR protein in breast cancer cells. These results provide more evidence that SAHA may stimulate TRAIL DR5-CTSB crosstalk, influence the activity of downstream TOR signalling pathway mainly through the AKTs pathway, and initiate the autophagy of breast cancer cells.

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“…Autophagolysosome utilizes various proteases involved in the vacuole to degrade contained substances for the metabolic needs. 25–28 …”

Section: Discussionmentioning

confidence: 99%

TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

Han

Zhou

et al. 2017

Cell Death Discov.

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“…In contrast, this recent evidence indicates that the absence of Atg8 family LC3/GABARAP proteins could not prevent the formation of autophagosome during PINK1-PARK2-dependent mitophagy. However, this protein is important for autophagosome size and stimulates autolysosome formation in autophagy [ 71 ]. Autophagy is a selective degradative process which requires various autophagic receptor proteins.…”

Section: P62 and Autophagymentioning

confidence: 99%

Autophagic Regulation of p62 is Critical for Cancer Therapy

Islam

Sooro

Zhang

2018

IJMS

188

125

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Sequestosome1 (p62/SQSTM 1) is a multidomain protein that interacts with the autophagy machinery as a key adaptor of target cargo. It interacts with phagophores through the LC3-interacting (LIR) domain and with the ubiquitinated protein aggregates through the ubiquitin-associated domain (UBA) domain. It sequesters the target cargo into inclusion bodies by its PB1 domain. This protein is further the central hub that interacts with several key signaling proteins. Emerging evidence implicates p62 in the induction of multiple cellular oncogenic transformations. Indeed, p62 upregulation and/or reduced degradation have been implicated in tumor formation, cancer promotion as well as in resistance to therapy. It has been established that the process of autophagy regulates the levels of p62. Autophagy-dependent apoptotic activity of p62 is recently being reported. It is evident that p62 plays a critical role in both autophagy and apoptosis. Therefore in this review we discuss the role of p62 in autophagy, apoptosis and cancer through its different domains and outline the importance of modulating cellular levels of p62 in cancer therapeutics.

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“…Many facets of autophagy are regulated by covalent linkage of ubiquitin‐like proteins in the LC3/GABARAP family (hereafter, collectively termed LC3) to phosphatidylethanolamine via an ubiquitin‐like E1‐E2‐E3 conjugation cascade . A dazzling assortment of functions arise from LC3 recruiting numerous different proteins harboring “LC3 Interacting Regions” (LIRs, featured by the consensus motif [W/F/Y]xx[L/I/V]) to membranes .…”

Section: Introductionmentioning

confidence: 99%

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Qiu

Zheng

et al. 2017

Protein Science

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Members of the LC3/GABARAP family of ubiquitin-like proteins function during autophagy by serving as membrane linked protein-binding platforms. Their C-termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin-like fold domains bind "LIR" (LC3-Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B-binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.

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Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

Cited by 30 publications

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TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

Autophagic Regulation of p62 is Critical for Cancer Therapy

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

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