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. 2018;14(2):296-310.
doi: 10.1080/15548627.2017.1402990.

THANATOS: An Integrative Data Resource of Proteins and Post-Translational Modifications in the Regulation of Autophagy

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Free PMC article

THANATOS: An Integrative Data Resource of Proteins and Post-Translational Modifications in the Regulation of Autophagy

Wankun Deng et al. Autophagy. .
Free PMC article

Abstract

Macroautophagy/autophagy is a highly conserved process for degrading cytoplasmic contents, determines cell survival or death, and regulates the cellular homeostasis. Besides ATG proteins, numerous regulators together with various post-translational modifications (PTMs) are also involved in autophagy. In this work, we collected 4,237 experimentally identified proteins regulated in autophagy and cell death pathways from the literature. Then we computationally identified potential orthologs of known proteins, and developed a comprehensive database of The Autophagy, Necrosis, ApopTosis OrchestratorS (THANATOS, http://thanatos.biocuckoo.org ), containing 191,543 proteins potentially associated with autophagy and cell death pathways in 164 eukaryotes. We performed an evolutionary analysis of ATG genes, and observed that ATGs required for the autophagosome formation are highly conserved across eukaryotes. Further analyses revealed that known cancer genes and drug targets were overrepresented in human autophagy proteins, which were significantly associated in a number of signaling pathways and human diseases. By reconstructing a human kinase-substrate phosphorylation network for ATG proteins, our results confirmed that phosphorylation play a critical role in regulating autophagy. In total, we mapped 65,015 known sites of 11 types of PTMs to collected proteins, and revealed that all types of PTM substrates were enriched in human autophagy. In addition, we observed multiple types of PTM regulators such as protein kinases and ubiquitin E3 ligases or adaptors were significantly associated with human autophagy, and again the results emphasized the importance of PTM regulations in autophagy. We anticipated THANATOS can be a useful resource for further studies.

Keywords: ATG; autophagy; phosphorylation; post-translational modification; ubiquitin.

Figures

Figure 1.
Figure 1.
The collection and curation of proteins that were experimentally identified to be associated with autophagy and cell death pathways from the literature. (A) In this study, we used multiple keywords to search the PubMed search engine, and obtained a total of 4,237 known AT, AP and NE proteins. Using 3,882 known proteins from 8 model organisms, we computationally detected their potential orthologs in 164 eukaryotes, and further performed an evolutionary analysis of ATG genes. Also, we carried out the enrichment analysis and the cancer mutation analysis for known human AT proteins, while the PTM analysis was conducted for known AT proteins in model species. Finally, we combined both known and computationally identified AT, AP and NE proteins together and developed the THANATOS database. (B) Based on experimental evidence, we annotated each known protein with a “+” or “-” to distinguish the positive or negative regulation in autophagy or PCDs. For 3,882 known AT, AP and NE proteins of 8 organisms, the proteins annotated only with “+” (only +) or “-” (only -), and with both “+” and “-” (+/-) were separately present. (C) The overlap of different types of known proteins for 8 model species. (D) The comparison of curated proteins from the literature between THANATOS and other existing resources. i. All, the number of nonredundant proteins in 8 public databases.
Figure 2.
Figure 2.
The distribution of experimentally and computationally identified AT, AP and NE proteins across 164 eukaryotes in THANATOS database. In our results, there were only 1,909, 1,134, 340, 39, 153, 77, 189 and 37 known AT, AP and/or NE proteins experimentally identified in H. sapiens, M. musculus, R. norvegicus, D. rerio, D. melanogaster, C. elegans, S. cerevisiae and A. thaliana respectively, whereas the integrative data set in total contained 12,222 proteins for the 8 species, with a >2-fold increase (Table S2). In total, THANATOS contains 191,543 proteins potentially associated with autophagy cell death pathways.
Figure 3.
Figure 3.
The search options of THANATOS database. (A) Simple search. The THANATOS database can be queried with one or multiple keywords. (B) Advanced search. This option allows a more precise search that 2 terms combined with operators of “and”, “or” and “exclude” can be specified in 2 different fields. (C) Batch search. The option permits users to input multiple keywords such as accession numbers or gene names in a line-by-line format for querying the database. (D) BLAST search. The option was designed for searching the database with a protein sequence in FASTA format.
Figure 4.
Figure 4.
The evolutionary conservation of 41 ATG genes across 164 species. ATG39, ATG40 and ATG41 were exclusively found in the yeast S. cerevisiae, whereas ATG25 and ATG30, ATG35 and ATG37 are only encoded in the K. pastoris. We classified ATG11 and ATG17 into a single group due to the sequence similarity in more complex eukaryotes and the functional similarity in autophagy. Based on the sequence and functional similarity, we also classified ATG3 and ATG10, ATG18 and ATG21, as well as ATG20 and SNX4/ATG24 into 3 groups, respectively. Clearly, there were 18 highly conserved ATG genes including ATG1 to ATG10, ATG11 and ATG17, ATG13, ATG16, ATG18 and ATG21, ATG20 and SNX4/ATG24. Their orthologs were detected in more than 85% (140) of 164 eukaryotes, and most of these ATG genes are involved in autophagosome formation.
Figure 5.
Figure 5.
The statistical enrichment analyses revealed that AT proteins are preferentially associated with human diseases, using the hypergeometric distribution. (A) AT proteins are significantly enriched in drug targets and cancer genes. (B) The KEGG-based enrichment analysis found that AT proteins are statistically over-represented in a number of cellular signaling and disease pathways. (C) There were 54 AT genes with a mutation frequency of ≥ 12% visualized for pancreatic adenocarcinoma. The PPI relations among these proteins are also present if available, 3 ATG genes, MAP1LC3A/LC3A, GABARAPL1 and MAP1LC3B/LC3B, are shown in pink. (D) The 54 mutated AT genes with a frequency of ≥ 5% were present for prostate cancer. Two ATG genes, ATG5 and WIPI1, are shown in pink. (E) A network of mutated AT proteins with at least one approved drug in pancreatic adenocarcinoma. The mutation frequency was shown in parentheses for each gene. The color indicates the mutation number of each gene detected in pancreatic adenocarcinoma samples from ICGC database. Genes with mutation frequency ≥ 8% were shown. (F) A drug-target network of mutated AT genes in prostate cancer. Genes with mutation frequency ≥ 5% were shown.
Figure 6.
Figure 6.
The phospho-regulation of human AT proteins. (A) The kinases and phosphatases were mapped from EKPD to AT proteins in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae and A. thaliana, respectively. The enrichment analysis was performed for each of the 7 species. Protein kinases were significantly enriched in most of the species, Except in C. elegans and A. thaliana, mainly due to the data limitation of known AT proteins in the 2 organisms. (B) The distribution of phosphorylated AT proteins and sites in 7 species. i. *, P value < 0.05; ii. **, P value < 0.01.
Figure 7.
Figure 7.
The phosphorylation networks among ATG proteins and their regulatory kinases for (A) H. sapiens, (B) M. musculus, and (C) S. cerevisiae. (D) The phosphorylation sites predicted kinases of human ULK1. The protein kinase family was shown as “group-family”. For example, AGC-DMPK refers to the family of dystrophia myotonica protein kinases (DMPKs) in the protein kinase A, G, and C (AGC) group. The detailed classifications of eukaryotic protein kinases can be accessed at EKPD (http://ekpd.biocuckoo.org/). pS, phospho-serine; pT, phospho-threonine.
Figure 8.
Figure 8.
Multiple PTMs are significantly associated with AT proteins. (A) The distribution of numbers of mapped substrates and sites of ubiquitination, acetylation, succinylation, sumoylation, methylation, glycation, propionylation, butyrylation, malonylation and phosphoglycerylation in 7 species. (B) The overlap of 4 major types of PTMs including phosphorylation, ubiquitination, acetylation and sumoylation for AT proteins. (C) The distribution and enrichment analysis of 11 PTMs mapped to human AT proteins. (D) The distribution of ubiquitin and ubiquitin-like enzymes mapped to human AT proteins. i. *, P value < 0.05; ii. **, P value < 0.01.

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Grant support

This work was supported by grants from the Special Project on Precision Medicine under the National Key R&D Program (2017YFC0906600), the National Basic Research Program (973 project) (2013CB933900), Natural Science Foundation of China (31671360), and International Science & Technology Cooperation Program of China (2014DFB30020).

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