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Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana . Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization.
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Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes
Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: β -grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the β -grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.
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- Award ID(s):
- 1710460
- PAR ID:
- 10108041
- Date Published:
- Journal Name:
- BioMed Research International
- Volume:
- 2018
- ISSN:
- 2314-6133
- Page Range / eLocation ID:
- 1 to 8
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1155/2018/6062520
