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Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations.

ABSTRACT: Autophagy is a conserved adaptive cellular pathway essential to maintain a variety of physiological functions. Core components of this machinery are the six human Atg8 orthologs that initiate formation of appropriate protein complexes. While these proteins are routinely used as indicators of autophagic flux, it is presently not possible to discern their individual biological functions due to our inability to predict specific binding partners. In our attempts towards determining downstream effector functions, we developed a computational pipeline to define structural determinants of human Atg8 family members that dictate functional diversity. We found a clear evolutionary separation between human LC3 and GABARAP subfamilies and also defined a novel sequence motif responsible for their specificity. By analyzing known protein structures, we observed that functional modules or microclusters reveal a pattern of intramolecular network, including distinct hydrogen bonding of key residues (F52/Y49; a subset of HP2) that may directly modulate their interaction preferences. Multiple molecular dynamics simulations were performed to characterize how these proteins interact with a common protein binding partner, PLEKHM1. Our analysis showed remarkable differences in binding modes via intrinsic protein dynamics, with PLEKHM1-bound GABARAP complexes showing less fluctuations and higher number of contacts. We further mapped 373 genomic variations and demonstrated that distinct cancer-related mutations are likely to lead to significant structural changes. Our findings present a quantitative framework to establish factors underlying exquisite specificity of human Atg8 proteins, and thus facilitate the design of precise modulators.Abbreviations: Atg: autophagy-related; ECs: evolutionary constraints; GABARAP: GABA type A receptor-associated protein; HsAtg8: human Atg8; HP: hydrophobic pocket; KBTBD6: kelch repeat and BTB domain containing 6; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MD: molecular dynamics; HIV-1 Nef: human immunodeficiency virus type 1 negative regulatory factor; PLEKHM1: pleckstrin homology and RUN domain containing M1; RMSD: root mean square deviation; SQSTM1/p62: sequestosome 1; WDFY3/ALFY: WD repeat and FYVE domain containing 3.

SUBMITTER: Jatana N

PROVIDER: S-EPMC6984608 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Myc-like regulatory factors carrying the basic helix-loop-helix (bHLH) domain belong to a large superfamily of transcriptional factors (TFs) present in all eukaryotic kingdoms. In plants, the representatives of this superfamily regulate diverse biological processes including growth and development as well as response to various stresses. As members of the regulatory MBW complexes, they participate in biosynthesis of flavonoids. In wheat, only one member (TaMyc1) of the Myc-like TFs family has been studied, while structural and functional organization of further members remained uncharacterized. From two Myc-subfamilies described recently in the genomes of Triticeae tribe species, we investigated thoroughly the members of the subfamily I which includes the TaMyc1 gene. RESULTS:Comparison of the promoter regions of the Myc subfamily I members in wheat suggested their division into two groups (likely homoeologous sets): TaMyc-1 (TaMyc-A1/TaMyc1, TaMyc-B1, TaMyc-D1) and TaMyc-2 (TaMyc-A2 and TaMyc-D2). It was demonstrated that the TaMyc-D1 copy has lost its functionality due to the frame shift mutation. The study of functional features of the other four copies suggested some of them to be involved in the biosynthesis of anthocyanins. In particular, TaMyc-B1 is assumed to be a co-regulator of the gene TaC1-A1 (encoding R2R3-Myb factor) in the MBW regulatory complex activating anthocyanin synthesis in wheat coleoptile. The mRNA levels of the TaMyc-A1, TaMyc-B1, TaMyc-A2 and TaMyc-D2 genes increased significantly in wheat seedlings exposed to osmotic stress. Salinity stress induced expression of TaMyc-B1 and TaMyc-A2, while TaMyc-A1 was repressed. CONCLUSIONS:The features of the structural and functional organization of the members of subfamily I of Myc-like TFs in wheat were determined. Myc-like co-regulator (TaMyc-B1) of anthocyanin synthesis in wheat coleoptile was described for the first time. The Myc-encoding genes presumably involved in response to drought and salinity were determined in wheat. The results obtained are important for further manipulations with Myc genes, aimed on increasing wheat adaptability.

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Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations.

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