Mixed lineage leukemia protein-1 (MLL1) is a member of the human SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which include MLL1-4 and SETd1A/B. Rearrangements of MLL1 are frequently present in acute leukemia, whereas genetic alterations in other SET1 family members are associated with developmental disorders as well as a number of cancers. A minimal evolutionarily conserved complex, which is formed by MLL1 and four additional proteins, is required for the sequential mono- and dimethylation of H3K4. WD40 repeat protein-5 (WDR5), one of the MLL1 core complex proteins, specifically interacts with a conserved WDR5 interaction motif of the SET1 proteins, also named the Win motif. Targeting the Win motif-WDR5 interaction with small-molecule drugs and Win-based peptidomimetics has emerged as a strategic approach for treatment of acute leukemia that harbors the MLL1 protein, because the MLL1-WDR5 interaction is a key regulatory mechanism of the MLL1 enzymatic activity. However, progress in identifying inhibitors of the MLL1-WDR5 interactions remains modest due to: (i) the lack of proteomics technologies for the quantitative evaluation of the transient protein-protein interactions (PPI) at the MLL1-WDR5 interface; (ii) the lack of a mechanistic knowledge pertaining to the MLL1-WDR5 recognition system. To address these scientific and technological gaps, we will develop monomeric protein-pore based sensors for sampling transient PPI at single-recognition event resolution. The central player of these sensors will be the t-FhuA protein pore, a heavily truncated derivative of ferric hydroxamate uptake component A (FhuA) of E. coli. t-FhuA will be fused to a water- soluble MLL1 SET binding domain via a short peptide tether. Such a MLL1 binding polypeptide-containing t-FhuA-based sensor will rely on precise protein engineering, along with biomolecular recognition, scalable high-resolution electrical recordings, and single-protein channel reconstitution. The presence of WDR5 will produce a specific, sensitive, and quantitative readout that encompasses reversible current blockades, the nature of which depends on the PPI strength and WDR5 concentration. The expected immediate outcomes will be the following: (i) the design, creation, and optimization of the next-generation t-FhuA-based sensors equipped with single receptor elements for the real-time, selective sampling of transient PPI in aqueous phase; (ii) the development of a mechanistic and quantitative information on the Win motif-WDR5 interactions for each SET1 family member; (iii) the multiplexed screening of Win motif- based inhibitors with improved translational potential. These research studies will ultimately lead to a fundamental basis for accelerated discoveries in clinical molecular diagnostics, proteomics, and biosensor technology.

Public Health Relevance

Targeting the MLL-WDR5 interactions shows prospects as a treatment strategy for several malignancies. However, progress has been limited by the lack of suitable technologies for the quantitative evaluation of these interactions. In response to this pressing demand, these studies are aimed at creating and optimizing a sensitive, specific, and modular biosensing platform for the examination of the MLL-WDR5 recognition system as well as rapid assessment of the Win motif-based inhibitors. !

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM129429-02S1
Application #
9940917
Study Section
Program Officer
Smith, Ward
Project Start
2018-06-01
Project End
2022-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Syracuse University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
002257350
City
Syracuse
State
NY
Country
United States
Zip Code
13244
Thakur, Avinash Kumar; Movileanu, Liviu (2018) Real-time measurement of protein-protein interactions at single-molecule resolution using a biological nanopore. Nat Biotechnol :
Wolfe, Aaron J; Gugel, Jack F; Chen, Min et al. (2018) Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics. J Phys Chem B 122:9471-9481