Biology subjects

MacKenzie, K. R.

Publications and source records attributed to MacKenzie, K. R..

4 recordsLinked to original sources

bioRxiv2026

Reversible in vivo regulation of drug metabolizing enzyme CYP1A2 activity through a dTAG knock-in strategy

Drug-metabolizing enzymes determine therapeutic exposure, efficacy and toxicity, but defining their isoform-specific functions in vivo remains challenging. Cytochrome P450 enzymes (P450s) are central to drug metabolism and pharmacokinetics (DMPK) and mediate the phase I metabolism of [~]75% of all marketed drugs. However, conventional knockout models can induce develop-mental and compensatory adaptations, and selective inhibitors are unavailable for many P450 isoforms. Here, we report the use of an inducible chemical-genetic platform for acute and specific degradation of the endogenous P450 enzyme Cyp1a2 in mice. Using CRISPR-Cas9-mediated knock-in editing, we introduced an FKBP12F36V degron into the endogenous Cyp1a2 locus to generate Cyp1a2dTAG mice. Treatment with the dTAG degrader dTAG-13 recruited an E3 ubiquitin ligase to CYP1A2dTAG, resulting in rapid and reversible proteasomal depletion of CYP1A2dTAG in vivo. Temporally controlled CYP1A2dTAG loss altered caffeine pharmacokinetics as expected, validating this model as a functional tool for DMPK studies. By enabling reversible suppression of drug-metabolizing enzymes without permanent deletion or chronic inhibitor exposure, this work establishes targeted protein degradation as a broadly adaptable strategy for studying drug metabolism in vivo and provides a foundation for extending inducible DMPK control to other P450s, conjugating enzymes and transporters.

pharmacology and toxicology↗

bioRxiv2022

An auto-inhibited state of protein kinase G and implications for selective activation

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Campaigns targeting nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis suggest that understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG I and I{beta} that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is still not well understood. Here we present a crystal structure of PKG I{beta} in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG I{beta} auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG I{beta} auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and activation of PKGs.

biochemistry↗

bioRxiv2020

Oleic acid triggers hippocampal neurogenesis by binding to TLX/NR2E1

Adult hippocampal neurogenesis underpins learning, memory, and mood, but diminishes with age and illness. The orphan nuclear receptor TLX/NR2E1 is known to regulate neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is unknown. We found that neural stem and progenitor cells require monounsaturated fatty acids to survive and proliferate. Specifically, oleic acid (18:1{omega}9) binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell cycle and neurogenesis genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation. These findings pave the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.

neuroscience↗

bioRxiv2017

The N-terminal domain of ALS-linked TDP-43 assembles without misfolding

TDP-43 forms inclusions in several neurodegenerative diseases, and both its N- and C-terminal domains are implicated in this process. We show that the folded TDP-43 N-terminal domain oligomerizes under physiological conditions and propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.

biophysics↗