Questions about a product, technology or experimental design? Give us a call 610-644-8845
Questions about a product, technology or experimental design? Give us a call 610-644-8845.
The functional consequences of protein polyubiquitination are determined by the type of ubiquitin chain assembled on the substrate. Among the eight known linkage types, lysine 48 (K48)-linked chains target proteins for proteasomal degradation, whereas lysine 63 (K63)-linked chains primarily regulate intracellular signaling, trafficking, autophagy, mitophagy, and mitochondrial biogenesis. Therapeutic strategies that exploit the ubiquitin–proteasome system (UPS) such as PROTACs (Proteolysis Targeting Chimeras) and molecular glues (MGs) harness E3 ligases to induce targeted protein degradation. However, current tools to measure endogenous ubiquitination with linkage specificity, such as mass spectrometry or chain-selective antibodies, are challenging and often lack sensitivity or throughput.
To overcome these limitations, we employed linkage-selective Tandem Ubiquitin Binding Entities (TUBEs) with sub-nanomolar affinity for specific polyubiquitin chain types, adapted to a microtiter plate format for high-throughput applications. Using RIPK2, a key mediator in inflammatory signaling, we demonstrated the utility of this platform. Upon stimulation with L18-MDP, RIPK2 underwent K63-linked ubiquitination, consistent with its role in NF-κB signaling. In contrast, treatment with a RIPK2-directed PROTAC (RIPK degrader-2) induced K48-linked ubiquitination, marking RIPK2 for proteasomal degradation.
Our results reveal that chain-selective TUBEs can effectively discriminate between linkage-specific ubiquitination events on endogenous proteins. Furthermore, we show that compounds promoting autophagy or mitophagy increase K63-linked polyubiquitination, supporting a mechanistic link between ubiquitin signaling and lysosomal degradation pathways. This chain-selective ubiquitin profiling platform provides a powerful, scalable method for drug discovery and mechanistic interrogation of the UPS and autophagic systems, enabling the identification of small molecules that act through distinct degradation pathways.
This poster utilizes a number of LifeSensors technologies and products: PA480, our K48 Linkage Elisa Kit our PA630 our K63 Linkage Elisa Kit, our PA770 our PROTAC In vitro Ubiquitination Assay Kit. LifeSensors inhibitors, SI9619, SI9649 and SI9710, are all key inhibitors for properly conducting this assay.
To utilize this technology for your own assay, whether to discover a novel E3 ligase, evaluate an engineered PROTAC, or screen for molecular glues, we suggest checking out our guide on optimization of our PA770 related kits.
To read further on this assay, please take advantage of Dr. Muhammed Shahzad Ali’s paper, in Nature Reports. The introduction can be found here, while the paper in full is listed here.
This technology will also be presented in detail at Discovery on Target 2025 by Dr. Shahzad Ali.
For any questions regarding this technology or how it can be adapted to your own assay, please don’t hesitate to reach out to our business office at BD@LifeSensors.com or drop us a direct question on our contact us page.
Best regards,
Your LifeSensors Team.
With ubiquitin’s core functions in plants and animals being so similar it has long been speculated that insights into the
The functional consequences of protein polyubiquitination are determined by the type of ubiquitin chain assembled on the substrate. Among the
While direct K-RAS inhibitors represent a major therapeutic advance, targeting this oncoprotein remains a significant challenge due to limited druggable
Pan-selective Tandem Ubiquitin-Binding Entities (TUBEs) are engineered, high-affinity reagents composed of multiple ubiquitin-associated (UBA) domains that bind polyubiquitin chains with
LifeSensors is pleased to bring attention to some of our academic colleagues who have used our products in the past
Different lysines on ubiquitin are involved in forming different ubiquitin chains, with K48-linked chains primarily signaling for proteasomal degradation, while
With ubiquitin’s core functions in plants and animals being so similar it has long been speculated that insights into the
The functional consequences of protein polyubiquitination are determined by the type of ubiquitin chain assembled on the substrate. Among the
While direct K-RAS inhibitors represent a major therapeutic advance, targeting this oncoprotein remains a significant challenge due to limited druggable
Pan-selective Tandem Ubiquitin-Binding Entities (TUBEs) are engineered, high-affinity reagents composed of multiple ubiquitin-associated (UBA) domains that bind polyubiquitin chains with
LifeSensors is pleased to bring attention to some of our academic colleagues who have used our products in the past
Different lysines on ubiquitin are involved in forming different ubiquitin chains, with K48-linked chains primarily signaling for proteasomal degradation, while
