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.

MicroRNAs (miRNAs) are short non-coding RNAs of 21–23 nucleotides that play important roles in virtually all biological pathways in mammals and in other multicellular organisms. miR-23a and miR-23b (miR-23a/b) are critical oncomiRs (miRNAs that are associated with human cancers) of gastric cancer, but their detailed roles in the initiation and progression of gastric cancer remain to be elucidated. In this study, we found that miR-23a/b were consistently upregulated in gastric cancer tissues. We then investigated the molecular mechanisms through which miR-23a/b contribute to gastric cancer and identified programmed cell death 4 (PDCD4) as a direct target gene of miR-23a/b. In contrast to the upregulated expression levels of miR-23a/b, PDCD4 protein levels were dramatically downregulated and inversely correlated with miR-23a/b in gastric cancer tissues. Moreover, we observed that cell apoptosis was increased by miR-23a/b inhibitors and decreased by miR-23a/b mimics in gastric cancer cells and that the restoration of PDCD4 expression attenuated the anti-apoptotic effects of miR-23a/b in gastric cancer cells, indicating that PDCD4 is a direct mediator of miR-23a/b functions. Finally, we showed that miR-23a/b significantly suppressed PDCD4 expression and enhanced tumor growth in a gastric cancer xenograft mouse model. Taken together, this study highlights an important role for miR-23a/b as oncomiRs in gastric cancer through the inhibition of PDCD4 translation. These findings may shed new light on the molecular mechanism of gastric carcinogenesis and provide a new avenue for gastric cancer treatment.

GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the γ2 subunit of GABAAR. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1–γ2-GABAAR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of γ2-GABAAR differentially modulate the receptor’s binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP–GABAAR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABAARs via modulating the trafficking of the receptor.