Autophagy is a process which is used to degrade cellular components and infectious agents, such as viruses, organelles and defective proteins. It is also activated during periods of bioenergetic stress in order to provide nutrients for cellular homeostasis. Previous blog entries have discussed how autophagy may be involved in TSC and VHL (see here and here respectively): this post will detail the work of Behrends et al. (2010), which has suggested that FNIP1 is a part of an autophagy interaction network.
In this study, 65 Flag-HA-tagged proteins associated with autophagy were retrovirally expressed in human embryonic kidney 293T cells, which were grown in standard conditions. Following immunoprecipitation, high-confidence candidate interacting proteins (HCIPs) were identified by mass spectroscopy using a Comparative Proteomic Analysis Software Suite (CompPASS). In total, 409 HCIPs were identified in what was referred to as an autophagy interaction network. From this analysis, it could be seen that FLCN and FNIP1 interacted with PRKAA2, which is a catalytic subunit of AMPK. This work ties in with the work of Baba et al. (2006) who first identified a link between FLCN, FNIP1 and AMPK. Moreover, Behrends et al. noted that FNIP1 also appeared to associate with Gamma-aminobutyric acid receptor-associated protein (GABARAP).
GABARAP is a member of the ATG8-family of proteins, which are necessary for autophagosomal development, and it is thought that they bind cargo through their LC3-interaction region docking site (LDS). However, in vitro binding of FNIP1 to GST-tagged GABARAP was unaffected when the LDS was mutated. In contrast, the TSC-associated autophagy substrate p62/SQSTM1 displayed reduced binding to the LDS-mutant GABARAP (when compared to the wild-type control), suggesting that there are both LDS-dependent and independent mechanisms for this interaction.
Using a U2OS cell line expressing a GFP-tagged GABARAP, siRNA knockdown of FNIP1 led to an increase in autophagosome production. However, this perceived increase could reflect an accumulation of autophagosomes due to a block in a later step of the pathway. Consequently, more work is necessary in order to understand the exact role of the FNIP1-GABARAP interaction during autophagy.
Considering the recent papers suggesting that FNIP1 is required for B-cell development (as discussed in two recent blog posts here and here), could autophagy be involved in this process? In addition, since FLCN interacts with FNIP1, could a dysregulation of autophagy play a role in the development of BHD syndrome?
- Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
- Behrends C, Sowa ME, Gygi SP, & Harper JW (2010). Network organization of the human autophagy system. Nature, 466 (7302), 68-76 PMID: 20562859