Mutant Rrm2 Protein Resists Proteasome Degradation
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Solution Overview
Problem
The variability in expression of the Rrm2 subunit of ribonucleotide reductase (RNR) in different cardiac diseases and disease models attenuates the efficacy of overexpressing the RNR subunits Rrm1 and/or Rrm2 to elevate dATP levels.
Innovation Solution
Introduction of mutant Rrm2 proteins that are resistant to proteasome-mediated degradation, thereby increasing their stability and leading to higher levels of RNR activity and dATP in cardiomyocytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If wild-type Rrm2 subunit is overexpressed to increase dATP levels, then ribonucleotide reductase activity increases, but the expression variability and proteasome-mediated degradation reduce the stability and efficacy of dATP elevation
Solution Approach 1:
The patent introduces a destabilizing element (degron) that makes the Rrm2 protein intentionally short-lived and subject to proteasome degradation. This controlled instability allows for dynamic control of protein levels and activity, enabling rapid response to cellular needs while maintaining overall system stability through regulated turnover rather than persistent expression
Solution Approach 2:
The patent modifies the Rrm2 protein by introducing specific amino acid mutations (e.g., at positions 30, 31, 32, 49, 51) that alter its interaction with the proteasome system. These parameter changes in the protein sequence directly affect degradation rates, allowing tuning of protein stability and activity levels to achieve optimal dATP production while reducing expression variability
2Quantity of substance
If proteasome activity is inhibited to increase Rrm2 expression, then Rrm2 levels increase, but this approach requires pharmacological intervention and may have off-target effects
Solution Approach 1:
The patent extracts and modifies the degradation signal (degron) within the Rrm2 protein sequence. By specifically targeting and altering the ubiquitin-binding degron region, the invention removes the need for external pharmacological proteasome inhibitors, achieving controlled Rrm2 expression through intrinsic protein design rather than external chemical intervention
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutant Rrm2 proteins result in higher and more stable RNR enzyme activity and increased deoxynucleotide levels in cardiomyocytes, enhancing cardiac muscle performance.
Implementation Method 1
Pharmacological inhibition of proteasome activity led to increased expression of Rrm2 in virally-transduced cardiomyocytes in vitro. Mutant Rrm2 proteins modified to interfere with proteasome-mediated degradation are described herein.
Implementation Method 2
Described herein are improved Rrm2 polypeptides that are resistant to degradation through the ubiquitin-proteasome pathway
Data Source
AI summary
Provided herein are isolated nucleic acids that encode a stable form of Rrm2 for the use of increasing the intracellular Rrm2 protein levels and cytosolic 2-deoxy-ATP (dATP) levels. Further provided herein are methods for treating a cardiac disease or disorder, e.g., myocardial infarction or myocardial ischemia, by administering the isolated nucleic acids, a polypeptide encoded by the isolated nucleic acids, or composition comprising the isolated nucleic acids to a subject in need thereof.