# How ubiquitin-carrying enzymes contribute to ubiquitin ligase specificity

> **NIH NIH R01** · UNIVERSITY OF NEVADA LAS VEGAS · 2021 · $460,026

## Abstract

The reversible control of enzyme activity is one of the cornerstone features enabling life. Cycles of protein
phosophorylation and de-phosphorylation have been appreciated for decades to regulate enzymes, and the
pharmacological inhibition of protein kinases and phosphatases has furnished a biotech industry intent on
treating various human diseases for nearly as long. One major drawback to this strategy is that the enzymes that
regulate protein phosphorylation represent perhaps 5 % of the human proteome, such that the vast majority of
aberrant proteins responsible for human disease have remained undruggable. More recently, drugs have been
invented that induce proximity between a disease-causing protein and an enzyme called a ubiquitin ligase that
promotes the destruction of the problematic protein. Indeed, this new drug modality is feeding a billion dollar per
year industry push to employ ubiquitin ligases to treat various human diseases including breast and prostate
cancers as well as multiple myeloma, to name a few. Most of these efforts have been utilizing a family of enzymes
called the Cullin-RING ligases (CRLs). With some 200 members in humans, the CRLs collectively control
approximately 20 % of ubiquitin-dependent protein degradation in cells. As such, an appreciation for how these
enzymes are regulated is of considerable interest to a wide audience from the scientific community. Similar to
the paradigm of protein phosphorylation, the control of CRLs is believed to be determined predominantly through
their reversible modification with a protein called NEDD8. And while human CRLs are known to partner with at
least 7 additional enzymes, which we refer to as ubiquitin-carrying enzymes (UCEs), that help promote CRL-
dependent protein substrate degradation, it also was believed that UCEs act promiscuously towards CRLs which
would preclude CRL regulation at the level of CRL-UCE interaction. However, CRL-UCE specificity is strongly
implied by the recent structure of an active CRL. Preliminary results here indicate that CRL-UCE specificity
endows these pairs with exceptionally rapid rates of ubiquitin transfer and with the capability of producing CRL
substrates modified with unique poly-ubiquitin chain architectures, potentially providing an additional layer of
control of CRL function beyond neddylation. In consideration of these observations, this application seeks to test
the hypothesis that UCEs generally display specificity for CRL-substrate complexes, and that the biological
purpose of these specific CRL-UCE pairs is to both enhance the rates of ubiquitin transfer from UCEs to CRL
substrates as well as to uniquely code the poly-ubiquitin chain to promote outcomes including protein
degradation or localization. The proposed studies will explore an entirely novel area of CRL biology, the
specificity of UCEs for CRLs, utilizing proteomic and cell biological assays to complement a powerful, quantitative
kinetics platform. These studies will illumin...

## Key facts

- **NIH application ID:** 10180287
- **Project number:** 1R01GM141409-01
- **Recipient organization:** UNIVERSITY OF NEVADA LAS VEGAS
- **Principal Investigator:** Gary L. Kleiger
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $460,026
- **Award type:** 1
- **Project period:** 2021-05-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10180287

## Citation

> US National Institutes of Health, RePORTER application 10180287, How ubiquitin-carrying enzymes contribute to ubiquitin ligase specificity (1R01GM141409-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10180287. Licensed CC0.

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