Biology subjects

Egan, E. D.

Publications and source records attributed to Egan, E. D..

6 recordsLinked to original sources

bioRxiv2026

High-throughput biochemical phenotyping of SHP2 variants reveals molecular basis of diseases and allosteric drug inhibition

Interpreting clinical and functional consequences of genetic variants remains challenging due to limited quantitative biochemical data at scale. We applied high-throughput microfluidic enzyme kinetics to profile 190 clinical variants of SHP2, a protein tyrosine phosphatase linked to developmental disorders and cancers. Through >300,000 reaction progress curves, we derived kinetic and thermodynamic parameters quantifying variant effects on catalysis, autoinhibition, stability, phosphopeptide binding, and drug responses. This multidimensional dataset reveals that dysregulated autoinhibition, rather than altered stability or catalysis, predominantly determines SHP2-associated pathogenesis. Thermodynamic modeling reveals that clinical-stage allosteric inhibitors preferentially stabilize a previously underappreciated, partially active conformation over the fully inactive state, leading to variant-dependent drug responses. Our high-throughput biochemical framework establishes a general approach to decipher the biochemical logic connecting protein variants to clinical outcomes.

biochemistry↗

bioRxiv2026

Activating Ras-MAPK pathway variants drive hippocampal clonal competition in human epilepsy

Mesial (a.k.a., medial) temporal lobe epilepsy (MTLE) is the most common focal epilepsy1,2 and, in drug-resistant cases, is treated by surgical removal of the anterior temporal lobe, which often shows neuronal loss and gliosis consistent with hippocampal sclerosis (HS)2. MTLE with HS has minimal contribution from germline genetic variation3, and is associated with prior precipitating insults such as prolonged childhood seizures and head trauma4-6. Somatic variants in Ras-MAPK pathway genes were recently reported in a few MTLE surgical specimens7,8, but their prevalence, clinical relevance, and underlying biological mechanisms remain unknown. Targeted duplex sequencing of hippocampal DNA from 462 surgical resections revealed significant enrichment of deleterious somatic variants in MTLE versus controls, with >40% of MTLE specimens harboring activating Ras-MAPK variants in PTPN11, NF1, BRAF, KRAS, and twelve genes not previously associated with focal epilepsy. Eight Ras-MAPK genes showed positive clonal selection in MTLE. Increased somatic variant burden predicted worse surgical outcome. Somatic Ras-MAPK variants at ultra-low (<0.5%) variant allele fractions were associated with older seizure onset and HS pathology, supporting a late prenatal or postnatal origin. Ras-MAPK variants in MTLE were enriched in cells derived from hippocampal progenitors--neurons, astrocytes, oligodendrocytes--in line with the known neuronal hyperexcitability and seizures induced by Ras-MAPK overactivation9,10; in contrast, Alzheimer disease hippocampi exhibited microglial enrichment of Ras-MAPK variants, consistent with prior reports11. Single-nucleus RNA sequencing showed increased expression of Ras-MAPK genes in neurons and upregulation of pathways mediating neurogenesis and neural development in MTLE. Functional validation of novel, recurrent PTPN11 variants confirmed gain-of-function, while cellular modeling in induced pluripotent stem cells demonstrated proliferative/survival advantages for mutant cells in mosaic culture. Overall, our data suggest that somatic Ras-MAPK variants and acquired risk factors may converge on clonal competition in the hippocampus to modulate epilepsy risk.

genetics↗

bioRxiv2023

Temporal Dynamics and Stoichiometry in Notch Signaling - from Notch Synaptic Complex Formation to NICD Nuclear Entry

Mammalian Notch signaling occurs when binding of Delta or Jagged to Notch stimulates proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to regulate target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20 min after contact. Synapse formation preceded entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after [~]45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with unprecedented spatiotemporal precision.

cell biology↗

bioRxiv2022

A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

molecular biology↗

bioRxiv2022

Structural Basis for Selective Proteolysis of ADAM10 Substrates at Membrane-Proximal Sites

The endopeptidase ADAM10 is a critical catalyst for regulated proteolysis of key drivers of mammalian development and physiology, and for non-amyloidogenic cleavage of the Alzheimers precursor protein as the primary -secretase. ADAM10 function in vivo requires formation of a complex with a C8-tetraspanin protein, with different ADAM10-C8-tetraspanin complexes having distinct substrate selectivity, yet the basis for such selectivity remains elusive. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and positions the enzyme active site about 20 [A] from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying selective ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

biochemistry↗

bioRxiv2022

An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 (DLL4) with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, engages human and murine Notch receptors with 500- to 1000-fold increased affinity compared to wild-type human DLL4. DeltaMAX also potently activates human Notch in plate-bound, bead-bound, and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch activation in response to either Delta-like (DLL) or Jagged (Jag) ligands, highlighting its utility as both an agonist and antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in primary activated human T cells. Taken together, our data defines DeltaMAX as a versatile biotechnological tool for broad-spectrum activation or inhibition of Notch signaling.

bioengineering↗