Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity.
The Journal of biological chemistry
The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia (CNL). However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in CNL and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues - the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at wild-type threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain (IL-31Rα). Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.
View details for DOI 10.1016/j.jbc.2023.104755
View details for PubMedID 37116708
Chemodivergent C(sp3)-H and C(sp2)-H Cyanomethylation Using Engineered Carbene Transferases.
2023; 6 (2): 152-160
The ubiquity of C-H bonds presents an attractive opportunity to elaborate and build complexity in organic molecules. Methods for selective functionalization, however, often must differentiate among multiple chemically similar and, in some cases indistinguishable, C-H bonds. An advantage of enzymes is that they can be finely tuned using directed evolution to achieve control over divergent C-H functionalization pathways. Here, we demonstrate engineered enzymes that effect a new-to-nature C-H alkylation with unparalleled selectivity: two complementary carbene C-H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an α-cyanocarbene into the α-amino C(sp3)-H bonds or the ortho-arene C(sp2)-H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold (nine mutations, less than 2% of the sequence) were needed to adjust the enzyme's control over the site-selectivity of cyanomethylation. The X-ray crystal structure of the selective C(sp3)-H alkylase, P411-PFA, reveals an unprecedented helical disruption which alters the shape and electrostatics in the enzyme active site. Overall, this work demonstrates the advantages of enzymes as C-H functionalization catalysts for divergent molecular derivatization.
View details for DOI 10.1038/s41929-022-00908-x
View details for PubMedID 36875868
View details for PubMedCentralID PMC9983643