How CHIP Autoubiquitination Regulates Its Interaction with Hsp70

Protein quality control is essential for maintaining cellular function by regulating protein folding, activity, and degradation. Disruptions in this system can lead to the accumulation of misfolded proteins, contributing to diseases such as Alzheimer’s and Parkinson’s.

This study investigates how autoubiquitination of CHIP, an E3 ubiquitin ligase, influences its interaction with the chaperone protein Hsp70. Using site-directed mutagenesis and ubiquitination assays, different CHIP variants were generated and analyzed. Protein interactions were measured using Bio-Layer Interferometry (BLI).

Results showed that wild-type CHIP exhibited polyubiquitination, while the CHIP9R mutant showed no ubiquitination, confirming successful mutation design. These findings suggest that specific lysine residues are critical for CHIP function and may influence its interaction with Hsp70.

This work provides insight into how protein regulation mechanisms contribute to cellular health and disease.

Diagram illustrating the ubiquitination pathway in which the E1 enzyme activates ubiquitin, transfers it to the E2 enzyme, and the E3 ligase attaches ubiquitin to a target protein, leading to protein regulation or degradation.

Research Questions

How does monoubiquitination of CHIP affect its interaction with Hsp70?

How do specific ubiquitination sites influence CHIP function?

What role does CHIP autoubiquitination play in protein quality control?

Methods

To investigate these questions, the following experimental approach was used:

Site-directed mutagenesis was used to create different CHIP variants
Lysine residues were selectively mutated to control ubiquitination sites
Monoubiquitinated CHIP was generated using K-zero ubiquitin
SDS-PAGE was used to confirm protein expression and modification
Protein-protein interactions were analyzed using Bio-Layer Interferometry (BLI)

BLI is a real-time optical technique that measures how proteins bind and interact with each other using a biosensor.

To investigate how CHIP functions and interacts with Hsp70, the following experimental approach was used:

Simplified diagram of the CHIP protein showing its major domains, including the TPR domain involved in chaperone binding, a central linker region, and the U-box domain responsible for ubiquitin ligase activity.

Diagram illustrating Bio-Layer Interferometry (BLI), where a biosensor measures protein-protein interactions in real time by detecting changes in light interference as molecules bind and dissociate.

Results/Findings and Discussion

CHIPWT showed clear polyubiquitination within 15 minutes
CHIP9R showed no ubiquitination, confirming that the mutations were successful
These results validate that the experimental design effectively controlled ubiquitination

The differences between CHIPWT and CHIP9R suggest that specific lysine residues are essential for ubiquitination to occur. This supports the idea that autoubiquitination is a regulated process rather than random.

These findings suggest that specific lysine residues are essential for CHIP ubiquitination and that autoubiquitination is a regulated process. This likely plays a role in modulating CHIP’s interaction with Hsp70, which may ultimately impact protein degradation pathways and cellular homeostasis.

Graph showing ubiquitination activity of wild-type CHIP (CHIPWT) over time, demonstrating rapid formation of polyubiquitin chains within 15 minutes of the reaction.

Graph showing ubiquitination activity of the CHIP9R mutant over time, indicating no detectable ubiquitination even after extended reaction time.

Conclusions and Future Study

Conclusion

Mutations successfully altered CHIP ubiquitination behavior
CHIPWT and CHIP9R showed expected differences in ubiquitination
Autoubiquitination plays a key role in regulating CHIP function
Results support the hypothesis that ubiquitination affects CHIP interactions

Future Work
Generate additional CHIP variants to isolate individual ubiquitination sites
Perform more detailed BLI experiments to quantify binding differences
Investigate how these interactions affect protein degradation pathways
Explore potential implications in neurodegenerative disease models

These future studies could provide deeper insight into the role of CHIP in neurodegenerative disease pathways.

The following is an image of poster presented at the 2026 Undergraduate Research Forum

Acknowledgements

National Institutes of Health Grant R35 GM128595

Dr. Richard C. Page

Page Lab members

Miami University Department of Chemistry and Biochemistry

References

De Silva, A. R. I., Shrestha, S., & Page, R. C. (2023). Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting. Analytical Biochemistry, 679, 115296.

Kriegesmann, J., & Brik, A. (2023). Synthesis of ubiquitinated proteins for biochemical and functional analysis. Chemical Science, 14(37), 10025–10040.

NACE Career Readiness Competencies

Through my research experience, I developed key NACE competencies including critical thinking, communication, teamwork, and career and self-development. I applied critical thinking by analyzing differences in ubiquitination between CHIP variants and interpreting experimental results. My communication skills improved through presenting research in both a poster and digital format. I strengthened teamwork by collaborating with lab members throughout the research process. This experience also supported my career development by reinforcing my interest in medicine and motivating me to continue building my scientific skills.

Research Compliance Protocols

Followed all laboratory safety procedures and guidelines
Properly handled and disposed of biological and chemical materials
Maintained ethical research practices throughout the study
Accurately recorded and reported all experimental data

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