Biology subjects

Miller, L. J.

Publications and source records attributed to Miller, L. J..

4 recordsLinked to original sources

bioRxiv2022

Intrinsic catalytic properties of histone H3 lysine-9 methyltransferases preserve monomethylation levels under low S-adenosylmethionine

S-adenosylmethionine (SAM) is the methyl donor for site-specific methylation reactions on histone proteins, imparting key epigenetic information. During SAM-depleted conditions that can arise from dietary methionine restriction, lysine di- and tri-methylation are reduced while sites such as Histone-3 lysine-9 (H3K9) are actively maintained, allowing cells to restore higher-state methylation upon metabolic recovery. Here, we investigated if the intrinsic catalytic properties of H3K9 histone methyltransferases (HMTs) contribute to this epigenetic persistence. We employed systematic kinetic analyses and substrate binding assays using four recombinant H3K9 HMTs (i.e., EHMT1, EHMT2, SUV39H1, and SUV39H2). At both high and low (sub-saturating) [SAM], all HMTs displayed the highest catalytic efficiency (kcat/KM) for monomethylation compared to di- and trimethylation on H3 peptide substrates. The favored monomethylation reaction was also reflected in kcat values, apart from SUV39H2 which displayed a similar kcat regardless of substrate methylation state. Using differentially-methylated nucleosomes as substrates, kinetic analyses of EHMT1 and EHMT2 revealed similar catalytic preferences. Orthogonal binding assays revealed only small differences in substrate affinity across methylation states, suggesting that catalytic steps dictate the monomethylation preferences of EHMT1, EHMT2, and SUV39H1. To link in vitro catalytic rates with nuclear methylation dynamics, we built a mathematical model incorporating measured kinetic parameters and a time course of mass spectrometry-based H3K9 methylation measurements following cellular SAM depletion. The model revealed that the intrinsic kinetic constants of the catalytic domains could recapitulate in vivo observations. Together, these results suggest catalytic discrimination by H3K9 HMTs maintain nuclear H3K9me1, ensuring epigenetic persistence after metabolic stress.

biochemistry↗

bioRxiv2021

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins: insight into mechanisms of G protein selectivity

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs 5 helix displaying a conformation that arises from "unwinding" of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs. One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.

pharmacology and toxicology↗

bioRxiv2020

Differential GLP-1R binding and activation by peptide and non-peptide agonists

Peptide drugs targeting class B1 GPCRs can treat multiple diseases, however there remains substantial interest in the development of orally delivered non-peptide drugs. Here we reveal unexpected overlap between signalling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist, PF 06882961, and GLP-1 that was not observed for another compound, OWL-833. Both compounds are currently in clinical trials for treatment of type 2 diabetes. High resolution cryo-EM structures reveal the binding sites for PF-06882961 and GLP-1 substantially overlap, whereas OWL-833 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not OWL-833, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

pharmacology and toxicology↗

bioRxiv2018

Sensory Over-Responsivity: Parent Report, Direct Assessment Measures, and Neural Architecture

BackgroundSensory processing differences are common across neurodevelopmental disorders. Thus, reliable measures are needed to understand biologic underpinnings of these differences. This study aims to define a scoring methodology specific to tactile (TOR) and auditory (AOR) over-responsivity. Second, using MRI Diffusion Tensor Imaging, we seek to determine whether children with AOR show measurable differences in their white matter integrity.\n\nMethodsThis study includes children with AOR and TOR from a mixed neurodevelopmental disorders cohort including autism and sensory processing dysfunction (n= 176) as well as neurotypical children (n= 128). We established cut-off scores for over-responsivity using the parent report: Short Sensory Profile (SSP), and the direct assessment: Sensory Processing-Three Dimensions:Assessment (SP-3D:A). Group comparisons, based on AOR phenotype, were then conducted comparing the white matter fractional anisotropy in 23 regions of interest.\n\nResultsUsing the direct assessment, 31% of the children with neurodevelopmental disorders had AOR and 27% had TOR. The Inter-test-agreement between SSP and SP-3D:A for AOR was 65% and TOR was 50%. Children with AOR had three white matter tracts showing decreased fractional anisotropy relative to children without AOR.\n\nConclusionsThis study identified cut scores for AOR and TOR using the SSP parent report and SP-3D:A observation. A combination of questionnaire and direct observation measures should be used in clinical and research settings. The SSP parent report and SP-3D:A direct observation ratings overlapped moderately for sensory related behaviors. Based on these initial structural neuroimaging results, we suggest a putative neural network may contribute to AOR.

neuroscience↗