Structural and functional characterization of the role of acetylation on the interactions of the human Atg8-family proteins with the autophagy receptor TP53INP2/DOR

ABSTRACT

The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a β-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.

KEYWORDS:

• GABARAP
• LC3-interacting region (LIR)
• MAP1LC3A/LC3A
• nuclear/cytoplasmic shuttling sirtuin 1 (SIRT1)
• X-ray crystallography

Acknowledgements

We would like to thank Dr. Jason Chin for the vectors to express specifically acetylated proteins, Dr. Jimmy Dikeakos for the GFP-LC3B vector and Pascal Raymond for useful discussions. Research described in this work was carried out in part at the Macromolecular Diffraction Facility at Cornell High Energy Synchrotron Source (MacCHESS), which is supported by the NSF under grant DMR-1332208 and NIH/NIGMS under grant GM-103485 and at the Canadian Light Source (CLS) which is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Abbreviations

DAPI:	=	4′,6-diamidino-2-phenylindole;
DTT:	=	dithiothreitol;
EDTA:	=	ethylenediaminetetraacetic acid;
EP300/p300:	=	E1A binding protein p300;
FM:	=	full medium;
GABARAP:	=	GABA type A receptor-associated protein;
GABARAPL1:	=	GABA type A receptor associated protein like 1;
GABARAPL2:	=	GABA type A receptor associated protein like 2;
GFP:	=	green fluorescent protein;
HP:	=	hydrophobic pocket;
IPTG:	=	isopropyl-b-D-thiogalactopyranoside;
ITC:	=	isothermal titration colorimetry;
LIR:	=	LC3-interacting region;
MAP1LC3/LC3:	=	microtubule associated protein 1 light chain 3;
NMR:	=	nuclear magnetic resonance;
PE:	=	phosphatidylethanolamine;
RFP:	=	red fluorescent protein;
SIRT1:	=	sirtuin1;
ST:	=	starvation;
TEV:	=	tobacco etch virus;
TP53INP2/DOR:	=	tumor protein p53 inducible nuclear protein 2;
UBL:	=	ubiquitin like;
WDFY3/ALFY:	=	WD repeat and FYVE domain containing 3;
WT:	=	wild-type.

Data availability statement

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). The PDB Accession codes are: 8T31 for the TP53INP2LIR-GABARAP complex, 8T32 for the TP53INP2LIR-GABARAP[K48Ac] complex, 8T33 for the TP53INP2LIR-GABARAP[K46Ac] complex, 8T4T for the TP53INP2LIR-LC3A complex, 8T35 for the TP53INP2LIR-LC3A[K51Ac] complex, and 8T36 for the TP53INP2LIR-LC3A[K49Ac] complex.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2353443.

Additional information

Funding

The work was supported by the Canadian Institutes of Health Research [130414 and 179806]; Canadian Institutes of Health Research [166104]; Defense Threat Reduction Agency [HDTRA1-22-1-0009].