Engineered Mono-PARPs for Isoform-Specific Target Identification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The understanding of mono-ADP-ribosylation (MARylation) mediated by mono-PARP enzymes is hindered by the lack of specific tools to identify their targets and inhibitors, due to the inability to distinguish between MARylation and PARylation, and the complexity of mono-PARP family members forming complexes and playing redundant roles in signal transduction.
Innovation Solution
Engineered mono-PARPs using an NAD+ analogue orthogonal to wild-type PARPs, and recombinant proteins with mutated catalytic domains that can specifically catalyze the addition of small molecule compounds to protein targets, allowing for the identification of direct targets and inhibitors through chemical genetic strategies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional molecular biology approaches (deletion and overexpression assays) are used to identify PARP targets, then target identification can be achieved, but the process is painstaking and time-consuming
Solution Approach 1:
The patent introduces an engineered PARP enzyme as an intermediary tool that bridges the gap between NAD+ and target proteins. This engineered enzyme contains a mutated catalytic domain that can specifically recognize and bind to an orthogonal NAD+ analogue, enabling selective labeling of targets without requiring traditional time-consuming deletion and overexpression assays for each individual PARP family member
Solution Approach 2:
The patent applies parameter changes by mutating specific amino acid residues in the PARP catalytic domain (e.g., changing the ceiling lysine to alanine, or modifying the floor position residues). These parameter changes in the enzyme structure enable it to recognize a different substrate (orthogonal NAD+ analogue) while maintaining catalytic activity, thus accelerating target identification
2Measurement precision
If specific antibodies are used to detect PARylated proteins, then detection can be achieved, but no such antibodies exist for detecting MARylated proteins
Solution Approach 1:
The patent uses an orthogonal NAD+ analogue as an intermediary carrier that contains a clickable handle. When the engineered PARP catalyzes the transfer of this modified NAD+ to target proteins, the clickable handle remains attached to the ADP-ribose moiety, enabling subsequent detection through chemical genetics approaches rather than requiring traditional antibodies
Solution Approach 2:
The patent replaces the biological detection system (antibodies) with a chemical detection system. By incorporating a non-natural amino acid or clickable handle into the NAD+ analogue, the detection method shifts from immunological recognition to chemical labeling and imaging, enabling detection of MARylation where antibodies are unavailable
3Measurement precision
If chemical tools are used to study MARylation, then some detection is possible, but none of these methods can distinguish between MARylation and PARylation
Solution Approach 1:
The patent segments the PARP family into distinct functional groups by creating engineered variants with specific mutations. The engineered PARP contains a mutated catalytic domain that is orthogonal to wild-type PARPs, meaning it only recognizes the orthogonal NAD+ analogue and not the natural substrate. This segmentation allows selective study of MARylation without interference from PARylation
Solution Approach 2:
The patent applies local quality by introducing specific mutations at key positions in the catalytic domain (ceiling lysine, floor position residues) while leaving the rest of the enzyme structure intact. These localized changes confer substrate specificity for the orthogonal NAD+ analogue, enabling discrimination between MARylation and PARylation through selective chemical labeling
4Measurement precision
If engineered PARPs with mutated catalytic domains are used, then specific target identification is enabled, but the enzyme structure must be modified
Solution Approach 1:
The patent segments the catalytic domain into functionally distinct regions: the ceiling position (residue 903 in PARP1) that contacts the nicotinamide ring, and the floor position (residues 1109-1112) that binds the adenosine moiety. By making targeted mutations in these segmented regions, the enzyme gains orthogonality while maintaining overall structural integrity and catalytic function
Solution Approach 2:
The patent applies parameter changes by systematically mutating specific amino acid residues at defined positions in the catalytic domain. These parameter changes (amino acid substitutions) alter the enzyme's substrate recognition properties without completely redesigning the enzyme structure, balancing engineering complexity with functional improvement
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
This approach enables the identification of isoform-specific targets for mono-PARPs, such as PARP10 and PARP11, revealing new biological roles and allowing for the decoupling of roles in signaling pathways, and the generation of databases to examine the biological roles of mono-PARPs in cells.
Implementation Method 1
The enzymes that catalyze ADP ribosylation, known as poly-ADP-ribose-polymerases (PARPs...) have been implicated in a number of physiological roles
Implementation Method 2
ADP-ribosylation—the transfer of the ADP-ribose (ADPr) moiety from nicotinamide adenine dinucleotide (NAD+) to amino acids in proteins—is a reversible posttranslational modification essential for cellular function in mammals
Data Source
AI summary
Mutant mono ADP-ribose-polymerases (mono-PARP) proteins and small molecule compound substrates specific for the mutant mono-PARP proteins as well as methods of using these compositions to identify protein targets of the mono-PARPs and to screen for antagonists of the mono-PARPs are described.