Notch ankyrin domain: evolutionary rise of a thermodynamic sensor

Abstract

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner.

Succinate Dehydrogenase, Succinate, and Superoxides: A Genetic, Epigenetic, Metabolic, Environmental Explosive Crossroad

“…Accordingly, cellular meltome studies identified SDH subunits among the cell heat resistant proteins [68]. Clustering of heat-releasing mitochondria in cells allows for heat-requiring processes to take place [69]. With a melting point close to 60°C, SDH subunits have therefore the intrinsic capacity to work stably in a hot mitochondrial background reaching 50°C; a temperature experimentally verified in a growing series of cell lines [70].…”

Redox-Mediated Rewiring of Signalling Pathways: The Role of a Cellular Clock in Brain Health and Disease

“…In this manner, signals generated downstream to the Notch pathway align the enhanced anabolic activity to mitochondrial energetic output. This pro-anabolic burst is terminated by a mitochondrial heat flux which destabilises the Notch transcriptional complex switching off the signalling pathway in a temperature-dependent manner [122].…”

Succinate Dehydrogenase, Succinate, and Superoxides: A Genetic, Epigenetic, Metabolic, Environmental Explosive Crossroad

“…Accordingly, cellular meltome studies identified SDH subunits among the cell heat-resistant proteins [ 72 ]. The clustering of heat-releasing mitochondria in cells allows for heat-requiring processes to take place [ 73 ]. With a melting point close to 60 °C, SDH subunits have therefore the intrinsic capacity to work stably in a hot mitochondrial background reaching 50 °C; a temperature experimentally verified in a growing series of cell lines [ 74 ].…”