Biology subjects

Grey, G.

Publications and source records attributed to Grey, G..

3 recordsLinked to original sources

bioRxiv2025

A basic leucine zipper uses a dimer pathway to locate its targets in DNA mixtures

The operation of Eukaryotic transcription factors remains enigmatic. Cyclic AMP-responsive element-binding protein (CREB) is a member of the basic zipper family, a superfamily of transcription factors which operate exclusively in eukaryotes and bind DNA targets as homodimers or heterodimers. Modulation of oligomerization provides an additional opportunity for transcriptional control by this (and similar) families over monomeric transcription factors. However, when dimerization occurs - before or after target binding - is not known. We performed a suite of in vitro stopped-flow kinetic measurements, including CREB basic zippers target search amongst excess non-target DNA. The extensive dataset enabled a kinetic and thermodynamic understanding of DNA binding that demonstrated most productive search is performed by dimeric, rather than monomeric, CREB. Equilibrium is approached very rapidly under physiologically relevant concentrations, where relative flux through the monomer pathway is only around 1 in every 10,000 complexes formed. This preference of mechanism is driven by CREB monomer having a substantially higher affinity for another CREB monomer than for its DNA target. Equilibrium experiments with eight other monomeric peptides further suggest this as a common feature amongst the bZIP proteins, with only one peptide (Jun) displaying similar affinities for both. The work has implications for understanding the nature of DNA target search, as well as designing efficient artificial transcription factors and transcriptional inhibitors.

biophysics↗

bioRxiv2025

Reverse remodelling of the mitochondria and cytoskeleton after respiratory heart rate variability pacing of the failing sheep heart

We have previously demonstrated that pacing the failing sheep heart with respiratory heart rate variability (RespHRV), a natural variability in the heart rate that is linked to respiration on a breath-by-breath basis, improves cardiac output dramatically. In this study, we used proteomics and super-resolution microscopy to explore the role of energetics and T-tubule cellular remodelling in response to ResPHRV pacing in an ischaemic ovine model of heart failure (HF). After 2 weeks of RespHRV pacing, cardiac output improved by 1.1 {+/-} 0.2 L/min (**p=0.003). Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) was used to probe differences between three groups: HF without any intervention, HF with RespHRV pacing and a healthy control group. Orthogonal Partial Least Squares (OPLS) discriminant analysis demonstrated a clear separation of all three groups by T score (***p<0.001) with the HF+RespHRV pacing group placed intermediate between the HF and control groups. The top 50 proteins negatively correlated with T score (down in HF, restored after RespHRV) were dominated by mitochondrial proteins, as confirmed by Pathway Enrichment Analysis (***p<0.001). Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) analysis confirmed this finding in selected targets (ACAA2, ACADS, CRAT, NDUFA8, and SUCLG1, *p<0.05). STimulated Emission Depletion (STED) microscopy identified a disruption of mitochondria structure in HF (*p<0.05) that was restored in the HF+R group (p=0.051). The area of mitochondria labelling was increased in the HF+RespHRV group compared to HF (**p=0.005). Many cytoskeletal proteins linked to mitochondria regulation and T-tubule remodelling were upregulated in HF and were reduced by RespHRV. MRM-MS was able to confirm these findings for selected targets (ANAXA2, CAVIN2, SPTBN1, TUBA4A). STED microscopy of collagen VI and the ryanodine receptor revealed cellular hypertrophy and remodelling of the T-tubules and cardiac junctions in HF sheep (*p<0.05), RespHRV showed a trend for reversing these structural changes. These data support the hypothesis that within the first two weeks of RespHRV pacing, there is an increase in mitochondrial repair and function coupled with re-organisation of the cellular cytoskeleton, which is consistent with the improvement in cardiac pump function.

cell biology↗

bioRxiv2024

Molecular chaperone BiP controls activity of the ER stress sensor Ire1 through interactions with its oligomers

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

biophysics↗