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ULK1 and ULK2 Are Less Redundant Than Previously Thought

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ULK1 and ULK2 Are Less Redundant Than Previously Thought bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 ULK1 and ULK2 are less redundant than previously thought: 2 Computational analysis uncovers distinct regulation and 3 functions of these autophagy induction proteins 4 5 6 Amanda Demeter1,2, Mari Carmen Romero-Mulero1,3, Luca Csabai1,4, Márton Ölbei1,2, 7 Padhmanand Sudhakar1,2,5, Wilfried Haerty1, Tamás Korcsmáros 1,2,* 8 9 1Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ, UK 10 2Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, UK 11 3Faculty of Biology, University of Seville, Seville, 41012, Spain 12 4Eötvös Loránd University, Budapest, 1117, Hungary 13 5Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium. 14 15 *Corresponding author details: 16 17 Dr Tamás Korcsmáros 18 ORCID id: https://orcid.org/0000-0003-1717-996X 19 Email: [email protected] 20 Phone: 0044-1603450961 21 Fax: 0044-1603450021 22 Address: Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ, UK bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 23 Abstract 24 Macroautophagy, the degradation of cytoplasmic content by lysosomal fusion, is an 25 evolutionary conserved process promoting homeostasis and intracellular defence. 26 Macroautophagy is initiated primarily by a complex containing ULK1 or ULK2 (two 27 paralogs of the yeast Atg1 protein). Deletion of ULK1 is sufficient to interrupt autophagy, 28 while ULK2 seems expendable. To understand the differences between ULK1 and 29 ULK2, we compared the human ULK1 and ULK2 proteins and their regulation. Despite 30 the high similarity in their enzymatic domain, we found that ULK1 and ULK2 have major 31 differences in their post-translational and transcriptional regulators. We identified 18 32 ULK1-specific and 7 ULK2-specific protein motifs serving as different interaction 33 interfaces. We identified three ULK1-specific and one ULK2-specific transcription factor 34 binding sites, and eight sites shared by the regulatory region of both genes. Importantly, 35 we found that both their post-translational and transcriptional regulators are involved in 36 distinct biological processes - suggesting separate functions for ULK1 and ULK2. For 37 example, we found a condition-specific, opposite effect on apoptosis regulation for the 38 two ULK proteins. Given the importance of autophagy in diseases such as inflammatory 39 bowel disease and cancer, unravelling differences between ULK1 and ULK2 could lead 40 to a better understanding of how autophagy is dysregulated in diseased conditions. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 41 Introduction 42 Macroautophagy, hereafter referred to as autophagy, is a lysosome-dependent 43 intracellular metabolic process. Autophagy is highly conserved in all eukaryotic cells, 44 and contributes to maintaining energy homeostasis, generating nutrients following 45 starvation and is crucial in promoting cell survival. Autophagy is present in cells at a 46 basal level but it is also activated at a higher level as a response to stress1,2. 47 Upon autophagy induction, an isolation membrane (phagophore) sequesters a 48 small portion of the cytoplasm, including soluble materials and organelles, to form the 49 autophagosome. The autophagosome then fuses with the enzyme-containing lysosome 50 to become an autolysosome and degrades the materials contained within it1. As 51 observed first in yeast, induction of autophagy is governed by the induction complex 52 formed by Atg proteins: Atg1-Atg13-Atg17-Atg31-Atg29. In mammalian cells, the 53 complex is composed of Atg1 homologs (ULK1 or ULK2), the mammalian homolog of 54 Atg13 (ATG13), RB1-inducible coiled-coil 1 (RB1CC1/FIP200) and ATG1013,4,5. 55 Induction of autophagy is tightly regulated through the protein mechanistic target of 56 rapamycin complex 1 (mTORC1): whilst complex-associated, mTORC1 phosphorylates 57 ULK1/2 and ATG13, resulting in their inactivation. Nevertheless, when cells are treated 58 with rapamycin or starved of nutrients, mTORC1 becomes separated from the induction 59 complex, resulting in dephosphorylation of these proteins and consequent autophagy 60 induction6. Interestingly, this type of autophagy induction is specific to higher eukaryotes 61 and some of the protist, however, as we previously demonstrated, non-unikont protists 62 (such as Toxoplasma spp., Leishmania spp. and Plasmodium spp.) lack the Atg1 63 complex, and induce their autophagy in different ways7. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 64 65 Figure 1: Domain structure of the yeast Atg1 protein and its human homologs. All Atg1 homologs have an N- 66 terminal kinase domain, while only Atg1, ULK1 and ULK2 share the C-terminal DUF3543 domain. The domain 67 structure of the proteins were downloaded from the.Pfam website (http://pfam.xfam.org/30). 68 69 The most studied component of the induction complex is the yeast Atg1 and its 70 homologs. There are five Atg1 orthologs in the human genome: ULK1, ULK2, ULK3, 71 ULK4, and STK36, all codes proteins containing a kinase domain (Figure 1)2,8. Out of 72 these genes, the protein product of ULK1 and ULK2 shows high similarity along the 73 whole length of the protein (98% query cover but only 52% identity)10 and in their kinase 74 domain (100% query cover with 78.71% identity)2. ULK1 and ULK2 genes code for 75 serine/threonine protein kinases that consist of a conserved N-terminal kinase domain 76 (catalytic domain), a central serine-proline rich region (PS), and a C-terminal interacting 77 domain (CTD)9. ULK1 and ULK2 are best known to be involved in the induction of 78 autophagy10,11, and are often mentioned interchangeably, however, there is growing 79 evidence for functional differences. For instance, Seung-Hyun et al. found that while 80 both proteins have shared functions in autophagy, they have opposing roles in lipid 81 metabolism10. ULK1 and ULK2 were also shown to be part of different molecular 82 complexes, possibly resulting in having independent functions or mode of regulation. It 83 was specifically found that following amino acid starvation, in contrast to ULK1, binding 84 of ULK2 to membranes was increased12. In most cell lines studies, loss of ULK1 is 85 sufficient to interrupt autophagy, and ULK2 was thought to have a redundant function. 86 On the other hand, in mice studies, the knockout of both ULK1 and ULK2 was needed bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 87 to show the neonatal lethality, which was seen also upon the loss of other core 88 autophagy genes such as ATG5 and ATG713–15. During erythroid differentiation, ULK1, 89 but not ULK2 was found to be upregulated, and deletion of ULK1, hence autophagy 90 deficiency resulted in delayed clearance of ribosomes and mitochondria in 91 reticulocytes13. Lee and Tournier also showed that the function of ULK2 is more likely to 92 compensate for the loss of ULK1 but in a cell-type specific manner2. Some speculations 93 indicate a dominance of either of the two ULK proteins determined by differential tissue 94 expression levels15,16. 95 Prompted by the evidence for the different roles of ULK1 and ULK2, we aimed to 96 compare the two human paralogs on a systems-level. We show that ULK1 and ULK2 97 are indeed different in their protein binding motifs and in their promoter sequences, 98 harbouring binding sites for different transcription factors. They also have different 99 experimentally validated post-translational and transcriptional regulators and their 100 respective interactors are important in distinct biological processes. The balance 101 between these two homologs and their separate processes can therefore be crucial in 102 diseases, especially when ULK1, ULK2 or their interactors are differentially expressed. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.27.967901; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 103 Results 104 The duplication of the yeast Atg1 happened at the base of the Chordates resulting 105 in the most similar homologs, ULK1 and ULK2. The multiple sequence alignment of 106 the yeast Atg1 protein and its human orthologs shows that the human ULK1 and ULK2 107 are the most similar to each other (Figure 2). We obtained similar results with alignment 108 of the kinase domain and alignment of the complete amino-acid sequences.
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