NDLI: Novel Cul3 binding proteins function to remodel E3 ligase complexes

Content Provider	Springer Nature : BioMed Central
Author	Wimuttisuk, Wananit West, Mark Davidge, Brittney Yu, Kebing Salomon, Arthur Singer, Jeffrey D
Abstract	Background Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as l eucine-r ich r epeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a B TB-C ul3-R bx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of C ul3-bound proteins that contain either the L RR or W D40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.
Related Links	https://bmcmolcellbiol.biomedcentral.com/counter/pdf/10.1186/1471-2121-15-28.pdf
Ending Page	17
Page Count	17
Starting Page	1
File Format	HTM / HTML
ISSN	26618850
DOI	10.1186/1471-2121-15-28
Journal	BMC Molecular and Cell Biology
Issue Number	1
Volume Number	15
Language	English
Publisher	BioMed Central
Publisher Date	2014-07-10
Access Restriction	Open
Subject Keyword	Cell Biology Biological Microscopy Life Sciences Cullin3 Tandem-affinity purification BTB domain-containing protein BCR ubiquitin ligase complex Mass spectrometry E3 ubiquitin ligase Protein purification Ubiquitin Ubiquitin ligase
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology
Journal Impact Factor	2.4/2023
5-Year Journal Impact Factor	2.5/2023

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