Structural insights into the interaction of the conserved mammalian proteins GAPR-1 and Beclin 1, a key autophagy protein

Abstract

Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267-284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1-GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267-284 docked onto GAPR-1, built using the CABS-dock server, indicates that this peptide binds to this GAPR-1 groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267-284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.

shRNA Glut-1 inhibits cell viability, apoptosis and migration of laryngeal carcinoma HEp-2 cells through regulating Beclin-1-mediated autophagy

“…Previous studies indicated that autophagy activation has a dual effect on the occurrence of tumors [37]. In most cases, it was shown that activated autophagy of cancer cells can induce autophagic cell death, which may represent a promising approach for cancer treatment [19][20][21][22][23][24][25][26][27][28][29][30][31]. In the present study, we showed that Glut-1 knockdown evoked autophagy activity in HEp-2 cells leading to subsequent cell apoptosis, suggesting that Glut-1 may be a novel inducer of cell autophagy and useful for the treatment of laryngeal carcinoma.…”

“…Furthermore, activated ATG1 can form a protein complex through a series of ubiquitination-like ligation reactions with other autophagy-related factors, such as ATG3 and ATG5, in a manner dependent on the activity of phosphorylated Beclin-1 [43]. Beclin-1 is a key protein in the process of autophagy in mammalian cells and is a direct mediator of autophagy [20,28,29,43]. Beclin-1 protein complex mediates autophagosome formation at the start of autophagy [20,28,29,43].…”

“…Beclin-1 is a key protein in the process of autophagy in mammalian cells and is a direct mediator of autophagy [20,28,29,43]. Beclin-1 protein complex mediates autophagosome formation at the start of autophagy [20,28,29,43]. In this process, the free form of LC3 (LC3-I) in the cytoplasm forms LC3-II by binding to the phosphatidylethanolamine group and integrates into the autologous bilayer membrane structure to complete closure of the autophagosome [20,28,29,43].…”

“…Beclin-1 protein complex mediates autophagosome formation at the start of autophagy [20,28,29,43]. In this process, the free form of LC3 (LC3-I) in the cytoplasm forms LC3-II by binding to the phosphatidylethanolamine group and integrates into the autologous bilayer membrane structure to complete closure of the autophagosome [20,28,29,43]. In addition, the level of autophagy-related gene Beclin-1 expression in head and neck cancer tissues was shown to be significantly lower than that in adjacent tissues, indicating that Beclin-1-mediated autophagy participates in the progress of tumor formation [20].…”

“…Beclin-1 is a homolog of the yeast ATG6/Vps30 gene, which was first identified as a molecule mediating autophagy in mammals [26]. It plays a positive regulatory role in the process of autophagy [27,28]. During the process of autophagy, Beclin-1 and Class III PI3K kinase form a complex that promotes the formation of autophagic vacuoles [29].…”