Different lysines on ubiquitin are involved in forming different ubiquitin chains, with K48-linked chains primarily signaling for proteasomal degradation, while other linkages, such as K63-linked chains, and single ubiquitin molecules (monoubiquitination) act as non-degradative signals for processes like DNA repair, signal transduction, and trafficking. Due to the diverse signaling and the multiple different lysines (K6, K11, K27, K29, K33, K48, and K63) involved in ubiquitination leading to very different fates, separation and careful study of a target protein’s ubiquitination is necessary in order to determine whether or not a protein is being degraded, stabilized, or trafficked from one part of the cell to another.

This paper, published in Nature Scientific Reports, a peer-reviewed open-access scientific journal published by Nature Portfolio, describes new technology in order to rapidly study ubiquitination alongside traditional western blot methods.

In this paper, Ali et al., utilizes Tandem Ubiquitin Binding Entities (TUBEs) to study the lysine specific ubiquitination of RIPK2 in response to L18-MDP stimulation with and without the presence of Ponatinib, a known RIPK2 inhibitor. While the data is consistent with previous literature on the topic, where RIPK2 undergoes K63 ubiquitination due to L18-MDP stimulation, the improvement in assay design comes in a 96-well plate based format for studying this type of interaction on a lysine specific TUBE coated microplate. Operating with a nanomolar affinity, these plates open easier, faster, and replicable testing at significantly higher throughput than western blots. Not only did the K63 assay perform consistent with literature, but the K48 assay consistently did as well, differentiating itself from the K63 assay, with a far more modest performance. This not only shows the difference between the two plates, but highlights further the importance of studying lysine specific ubiquitination.

Introduction

Ubiquitination is a versatile and highly regulated post-translational modification that involves the addition of ubiquitin (8 kDa) to target proteins, influencing various cellular functions such as proteolysis, cell cycle, DNA repair, apoptosis, and immune responses The process of ubiquitination involves a cascade of enzymes (E1, E2, and E3) wherein the E3 ubiquitin ligases select the target protein for ubiquitination and degradation. There are over 600 E3 ligases encoded in the human genome of which only a small fraction of E3s have been functionally characterized. Dysregulation of E3 ligases often leads to the development of diseases such as central nervous system disorders, inflammation, metabolic dysfunctions, and several cancers. Ubiquitination can occur mainly through 8 different types of linkages (M1, K6, K11, K27, K29, K33,K48 and K63) among which the K48 and K63 linkages are most widely studied. The initial evidence suggesting distinct functions of different ubiquitin chain linkages came from studies involving yeast mutants with mutations in either K48 or K63 of ubiquitin, where K63-linked polyubiquitin was found to be involved in DNA repair while K48-linked polyubiquitin was crucial for protein degradation and cell cycle progression. These findings established the notion that specific chain linkages play unique roles in cellular processes. It is now well established that K48-linked chains target proteins for degradation, while K63-linked chains regulate protein function, signal transduction, subcellular localization, or protein-protein interactions. K63 ubiquitination is involved in the formation of signalosomes that activate downstream signaling molecules, particularly in NF-κB and MAPK pathways. NF-κB activation involves K63 ubiquitination of NF-κB essential modulator (NEMO), promoting IKK complex assembly and gene expression related to inflammation. K63 ubiquitination also contributes to the activation of the NLRP3 inflammasome, involved in pro-inflammatory cytokine production.

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