Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins

Abstract

The mammalian Atg8 family proteins are central drivers of autophagy and contain six members, classified into the LC3 and GABARAP subfamilies. Due to their high sequence similarity and consequent functional overlaps, it is difficult to delineate specific functions of Atg8 proteins in autophagy. Here we discover a super-strong GABARAP-selective inhibitory peptide harbored in 270/480 kDa ankyrin-G and a super-potent pan-Atg8 inhibitory peptide from 440 kDa ankyrin-B. Structural studies elucidate the mechanism governing the Atg8 binding potency and selectivity of the peptides, reveal a general Atg8-binding sequence motif, and allow development of a more GABARAP-selective inhibitory peptide. These peptides effectively blocked autophagy when expressed in cultured cells. Expression of these ankyrin-derived peptides in Caenorhabditis elegans also inhibited autophagy, causing accumulation of the p62 homolog SQST-1, delayed development and shortened life span. Thus, these genetically encodable autophagy inhibitory peptides can be used to occlude autophagy spatiotemporally in living animals.

Acetylation modulates LC3 stability and cargo recognition

“…On the inner membrane, this protein binds to cargo receptors to complete autophagic cargo recognition in selective autophagy [6]. It is well known that these interactions are highly dependent on a consensus LC3-interacting region (LIR, W/F/Y-X-X-L/I/V) in LC3 target proteins [7,8]. The hydrophobic residues in LIR bind to two hydrophobic pockets of LC3, and the former contains some well-conserved residues including the acetylation sites [3,9].…”

“…Human LC3 subfamily comprises three highly homologous isoforms, LC3A, LC3B, and LC3C [7]. The main purpose of this study was to examine the regulation role of acetylation on LC3B (a reliable autophagosome marker, hereafter referred to as LC3).…”

“…When on the outer membrane, LC3 guides autophagosome to lysosomes along microtubule by associating with FYCO1 [5]. It is well known that these interactions are highly dependent on a consensus LC3-interacting region (LIR, W/F/Y-X-X-L/I/V) in LC3 target proteins [7,8]. It is well known that these interactions are highly dependent on a consensus LC3-interacting region (LIR, W/F/Y-X-X-L/I/V) in LC3 target proteins [7,8].…”

SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

“…Firstly, the deficiency of an Ankyrin-repeat containing Drosophila protein was shown to rescue mitochondrial pathology and phenotypes triggered by PINK1 and Parkin mutations [118]. Secondly, peptides derived from ANK2 were observed to be potent and specific inhibitors of autophagy via ATG8-binding [119,120].…”

Structural basis for extremely strong binding affinity of giant ankyrins to LC3/GABARAP and its application in the inhibition of autophagy

“…The ANK3 LIR motif was found in the N-terminal region of the giant insertion that is predicted to be unstructured, but a molecular mechanism of this extremely strong LIR-based interaction remained obscure. Recently, Li et al embarked on elucidation of the structural basis of the ANK3 LIR motif and on development of autophagy inhibitors that take advantage of this interaction [3]. First, Li et al mapped the minimal GABARAP binding region of ANK3 to a 26-amino acid peptide (residues 1985-2010 in rat ANK3).…”