Biology subjects

Brown, S. H.

Publications and source records attributed to Brown, S. H..

4 recordsLinked to original sources

bioRxiv2024

Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS), a leading inherited cause of intellectual disability and autism, is frequently accompanied by sleep and circadian rhythm disturbances. In this study, we comprehensively characterized these disruptions and evaluated the therapeutic potential of a circadian-based intervention in the fragile X mental retardation 1 (FMR1) knockout (KO) mouse. The Fmr1 KO mice exhibited fragmented sleep, impaired locomotor rhythmicity, and attenuated behavioral responses to light, linked to an abnormal retinal innervation and reduction of light-evoked neuronal activation in the suprachiasmatic nucleus. Behavioral testing revealed significant deficits in social memory and increased repetitive behaviors in the mutants, which correlated with sleep fragmentation. Remarkably, a scheduled feeding paradigm (6-hour feeding/18-hour fasting) significantly enhanced circadian rhythmicity, consolidated sleep, and improved social deficits and repetitive behaviors in the Fmr1 KO mice. This intervention also normalized the elevated levels of some pro-inflammatory cytokines, including IL-12 and IFN-{gamma}, in the mutants blood, suggesting that its benefits extend to inflammatory pathways. These findings highlight the interplay between circadian disruption, behavior, and an inflammatory response in FXS, and provide compelling evidence that time-restricted feeding may serve as a promising non-pharmacological approach for improving core symptoms in neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

DET1 dynamics underlie co-operative ubiquitination by CRL4DET1 COP1 complexes

Ubiquitin ligases regulate core cellular processes through diverse mechanisms. The ubiquitin ligase COP1 is conserved from plants to humans and is particularly important for targeting developmental transcription factors for ubiquitination. COP1 can function independently, but can also be recruited to Cullin-4 ubiquitin ligase complexes via the DET1 adaptor protein. However, the mechanism of action of complexes containing COP1 and DET1 is not well understood. Here we report the cryo-electron microscopy structure of human DET1, bound to proteins that enable Cullin-4 recruitment (DDB1-DDA1) and an additional ubiquitin ligase enzyme (Ube2e2). We observe that DDA1 stabilises a closed conformation of DET1, binding adjacent to a unique Ube2e2 binding-insert in DET1. Moreover, we demonstrate that closure of DET1 underlies COP1 recruitment, which binds in an antiparallel dimeric state. Disrupting either the Ube2e2-binding insertion of DET1, or distinct recruitment sites on COP1, abolish DET1-COP1 binding and DET1-mediated modulation of COP1 levels. The multifaceted architecture provides an efficient platform for ubiquitination of substrates, or COP1 itself, by Cullin-4DET1 and offers multiple opportunities for physiological regulation.

biochemistry↗

bioRxiv2023

Highly amine-reactive graphene-oxide EM grids for biochemical surface modification in aqueous buffer

Graphene oxide (GO), an oxidized derivative of graphene, has found application in cryo-electron microscopy (cryo-EM) as a hydrophilic and transparent solid support on which to adsorb biological macromolecules, providing an alternative to traditional aqueous films. Current applications generally adsorb the macromolecule directly onto unmodified GO or modify the GO surface with polyethylene glycol-amine reagents. This nucleophilic amine reaction must be performed in an aprotic organic solvent and therefore precludes the use of biological samples such as nucleic acids and peptides. The utility of GO could be expanded by the ability to covalently modify its surface with biochemical affinity reagents such as small- molecule metabolites, peptides, or nucleic acids, in aqueous buffer at neutral pH. Presented here is a chemical procedure that converts all oxygen functionalities of GO to highly amine- reactive glycidyl epoxide groups, achieved without the need of specialized laboratory equipment. We show that single sheets of glycidyl epoxide-modified GO react on the EM grid with primary amines at micromolar concentrations in minutes at room temperature in aqueous buffer. Given the ease of derivatizing biochemical reagents with amines, the chemistry described here will enable imaging of macromolecules immobilized on GO through specific biochemical and biologically relevant binding interactions.

molecular biology↗

bioRxiv2021

Mechanism of transcription modulation by the transcription-repair coupling factor

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes distressed RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

biophysics↗