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Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by the free-interface diffusion method.

ABSTRACT: Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12-Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12-Atg5-Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12-Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12-Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12-Atg5-Atg16N ternary complex was formed by mixing purified Atg12-Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 A resolution.

SUBMITTER: Noda NN

PROVIDER: S-EPMC2394807 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 microL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 A resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays.

Project description:BIRC5/Survivin is known as a dual cellular functions protein that directly regulates both apoptosis and mitosis in embryonic cells during embryogenesis and in cancer cells during tumorigenesis and tumor metastasis. However, BIRC5 has seldom been demonstrated as a direct macroautophagy/autophagy regulator in cells. ATG7 expression and ATG12-ATG5-ATG16L1 complex formation are crucial for the phagophore elongation during autophagy in mammalian cells. In this study, we observed that the protein expression levels of BIRC5 and ATG7 were inversely correlated, whereas the expression levels of BIRC5 and SQSTM1/p62 were positively correlated in normal breast tissues and tumor tissues. Mechanistically, we found that BIRC5 negatively modulates the protein stability of ATG7 and physically binds to the ATG12-ATG5 conjugate, preventing the formation of the ATG12-ATG5-ATG16L1 protein complex in human cancer (MDA-MB-231, MCF7, and A549) and mouse embryonic fibroblast (MEF) cells. We also observed a concurrent physical dissociation between BIRC5 and ATG12-ATG5 (but not CASP3/caspase-3) and upregulation of autophagy in MDA-MB-231 and A549 cells under serum-deprived conditions. Importantly, despite the fact that upregulation of autophagy is widely thought to promote DNA repair in cells under genotoxic stress, we found that BIRC5 maintains DNA integrity through autophagy negative-modulations in both human cancer and MEF cells under non-stressed conditions. In conclusion, our study reveals a novel role of BIRC5 in cancer cells as a direct regulator of autophagy. BIRC5 may act as a "bridging molecule", which regulates the interplay between mitosis, apoptosis, and autophagy in embryonic and cancer cells.AbbreviationsACTA1: actin; ATG: autophagy related; BIRC: baculoviral inhibitor of apoptosis repeat-containing; BAF: bafilomycin A1; CQ: chloroquine; CASP3: caspase 3; HSPB1/Hsp27: heat shock protein family B (small) member 1/heat shock protein 27; IAPs: inhibitors of apoptosis proteins; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA.

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Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by the free-interface diffusion method.

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