Dominant mutations play a vital role in unveiling essential structure-function connections within proteins, especially when it comes to understanding the precise functions of multifunctional proteins at the residue level. These dominant mutations can often offer more valuable insights than loss-of-function variants either by acting through sequestering substrates at a particular reaction step or by forming mixed dysfunctional oligomers with WT subunits. It is imperative to develop high-throughput screening methods to analyze dominant variants, as they can serve as powerful tools for elucidating the disease-relevant, specific functions of multifunctional proteins. An insightful study was conducted to comprehensively investigate the prevalence of dominant negative variants in ubiquitin, a key regulator of cellular homeostasis, in a collaborative effort led by Dr. Parul Mishra and her team from the School of Life Sciences, University of Hyderabad along with Prof. Bolon from the University of Massachusetts Chan Medical School, USA.

Dr. Parul Mishra

The study involved high throughput fitness scanning of systematically generated 5100 mutants of ubiquitin in yeast. The researchers identified widespread occurrence of approximately 400 dominant negative mutations underscoring the profound impact of dominant variants in constraining the pace and trajectory of ubiquitin evolution. Notably, certain ubiquitin variants exhibited modified affinities to their pathway interactors, while others demonstrated the formation of unconventional polyubiquitin chains. This unbiased approach to protein engineering, geared toward identifying dominant mutations, holds significant promise in deciphering the precise structure-function relationship of other proteins as well.

This work was recently published in Cell Reports, a highly reputable Cell Press Journal. Article title “Systematic profiling of dominant ubiquitin variants reveals key functional nodes contributing to evolutionary selection”

By shedding light on the prevalence and implications of dominant mutations in a multifunctional protein ubiquitin, this research serves as a vital milestone in the ongoing quest to unravel the complexities of cellular regulatory mechanisms.

The team is thankful to the University of Hyderabad, Ministry of Education, Department of Biotechnology and Prime Minister’s Research Fellowship for supporting their research.