iASPP is a novel autophagy inhibitor in keratinocytes

Abstract

BSTRACTThe protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fissionfusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5-Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.

iLIR database: A web resource for LIR motif-containing proteins in eukaryotes

“…These proteins have been described to take part in a broad range of processes related to autophagy, such as immunity (NFKBIA/IκBα, 51 IRF1 [interferon regulatory factor 1], 52,53 PPP1R13L/iASPP, 54 EIF2AK2/PKR, 55 RELA/NF-κB-p65 56,57 ) or oncogenesis (BRCA1, 58,59 MYC, 60,61 RB1 [retinoblastoma 1], 62,63 TSC2/Tuberin, 64 FOXO1, 65 XIAP 66,67 ). A few posttranslational modification enzymes have also been identified, such as 2 ubiquitin ligases (HERC1 68,69 and XIAP 66,67 ), 4 kinases (PKD2/polycystin 2, 70,71 MARK4, 72 SIK2, 73 CAMKK2/CaMKKβ 74 ), one deacetylase (HDAC4 75 ), one methyltransferase (EHMT2/G9a 76,77 ) and one phosphatase (PTPN13/PTPL1 78 ) (Table S3).…”

Flow cytometric assays for the study of autophagy

“…438 However when the cells are much larger than those included in 439 this study such as keratinocyte cell lines in other published studies 440 were LysoTracker signals do match LC3B up-regulation [39]. 441 The technique of organelle mass quantification to determine 442 levels of mitophagy and ER elongation or phagy is a useful tech-443 nique in that it demonstrates that the same reagents can give dif- …”

“…Blotting can be used to quantitate the degree of autophagy by mea-121 suring the autophagy marker protein, LC3B [10][11][12]. LC3B can also LC3B levels detected in this study [39]. phase of the cell cycle (Fig.…”

“…37 Given that numerous autophagy inducing agents cause cell cycle arrest, the cell cycle phase distribution 38 was investigated and LC3B antigen was shown to increase as cells progressed through the cell cycle. 39 LysoTracker dyes have been previously employed to investigate the autophagic process and here the 40 LysoTracker signal increased in autophagic cells in relation to controls. Organelle autophagy of mitochon-41 dria and Endoplasmic Reticulum (ER), termed mitophagy and ER phagy was determined flow cytometri-42 cally by the employment of organelle mass probes, MitoTracker Green (MTG) and ER Tracker Green 43 (ERTG).…”

The Roles of Autophagy and the Inflammasome during Environmental Stress-Triggered Skin Inflammation

“…In addition, the ATG7-deleted keratinocytes irradiated by UVA showed defective clearance of p62 and the elevation of nuclear factor-like 2 (Nrf2) target gene expression [51]. Besides the canonical autophagy pathway, ATG5/ATG7-independent but Beclin1-dependent autophagy has been described in keratinocytes [52]. These studies suggested that the canonical and non-canonical autophagy pathways in keratinocytes participate in UVR-induced autophagy.…”