Receptor tyrosine kinases (RTKs) are a major class of cell surface receptors, important in cell signalling events associated with a variety of functions. High-throughput (HTP), coarse-grained molecular dynamics (CG-MD) simulations have been used to investigate the dimerization of the transmembrane (TM) domain of selected RTKs, including epidermal growth factor receptor (EGFR) and muscle-specific kinase (MuSK). EGFR activation requires not only a specific TM dimer interface, but also a proper orientation of its juxtamembrane (JM) domain. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is known to abolish EGFR phosphorylation through interaction with basic residues within the JM domain. Here, a multiscale approach was used to investigate anionic lipid clustering around the TM-JM junction and how such clustering is modulated by the mutation of basic residues. The simulations demonstrated that PIP₂ may help stabilize the JM-A antiparallel dimer, which may in turn help stabilize TM domain helix packing of the N-terminal dimerization motif. A proximal TM domain residue has been implicated in the inhibition of ganglioside GM3 in phase-separated membranes. Here, CG simulations were used to explore the dynamic behaviour of the EGFR TM domain dimer in GM3-containing and GM3-depleted bilayers designed to resemble lipid-disordered (Ld) and phase-separated (Ld/Lo) membranes. The simulations suggest that the presence of GM3 in Ld/Lo bilayers can disrupt and destabilize the TM dimer, which helps to explain why GM3 may favour monomeric EGFR in vivo. To gain insights into the dynamic nature of the intact EGFR, a nearly complete EGFR dimer was modelled using available structural data and embedded in an asymmetric compositional complex bilayer, which resembles the mammalian plasma membrane. The results demonstrated the dynamic nature of the EGFR ectodomain and its predicted interactions with lipids in the local bilayer. Strong protein-lipid interactions, as well as lipid-lipid interactions, affect the local clustering of lipids and the diffusion of lipids in the vicinity of the protein on both leaflets.

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Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

The epidermal growth factor receptor (EGFR) belongs to the erbB family of receptor tyrosine kinases which consists of four members (EGFR, ErbB2, ErbB3 and ErbB4). Upon ligand binding, the EGFR is capable of dimerization with other erbB receptors and propagates signals regulating a diverse array of cellular physiologies, including cell growth, migration and survival. Dysregulation of the EGFR is important for development and progression of different types of cancers, including breast cancer. Breast cancer is the second leading cause of cancer death in women. EGFR overexpression has been observed in about 15% of all breast cancers. Moreover, in triple negative breast cancer (TNBC), which is a more aggressive type of breast cancer and lacks effective therapies, up to 50% of tumors are found to overexpress EGFR. Targeted therapy against EGFR has been used in TNBC. However, limited efficacy has been observed in TNBC due to intrinsic and acquired resistant mechanisms. In order to overcome this issue, we have developed two novel therapeutic peptides derived from the nuclear localization signal (NLS) sequence and juxtamembrane domain of EGFR and investigated their efficacy in regard to inhibiting EGFR translocation and activation in TNBC. EGFR has been found to translocate into the nucleus and nuclear EGFR can affect gene transcription, cell proliferation, stress response and DNA repair through interacting with different components in the nucleus. Importantly, these functions of nuclear EGFR correlate with cancer prognosis and therapeutic resistance. We found that an EGFR NLS-derived peptide (ENLS peptide) could inhibit activated EGFR (pY845) undergoing nuclear translocation. We also showed that this ENLS peptide sensitized breast cancer cells to AG1478 (EGFR tyrosine kinase inhibitor) treatment. The juxtamembrane domain of EGFR regulates its trafficking to the nucleus and mitochondria, interaction with calmodulin and calcium signaling, and participates in dimerization and activation of EGFR. These non-traditional kinase related functions of EGFR represent a novel target for EGFR therapy. We found that a mimetic peptide of the juxtamembrane domain of EGFR (EJ1 peptide) could effectively inhibit EGFR activation through promoting inactive dimer formation. It could also effectively kill cancer cells through processes of apoptosis and necrosis. Mechanistically, this EJ1 peptide affects membrane integrity thereby leading to calcium influx, disruption of mitochondrial membrane potential and reactive oxygen species (ROS) accumulation. Importantly, EJ1 peptide appeared to be effective in inhibition of tumor growth and metastasis in a transgenic mouse model of breast cancer and showed no observable toxicity. ErbB3, another member of the erbB family, represents an important driver of the parallel signaling pathway to EGFR as well as a key regulator of PI3K/AKT activity which is important for therapeutic resistance. ErbB3 has been shown to interact with MUC1. The interaction between MUC1 and EGFR promotes EGFR stability through recycling of receptors. We found that MUC1 expression also affected ErbB3 activity and stability through ErbB3/EGFR/MUC1 complex formation. In conclusion, we demonstrated that two EGFR-derived peptides, working through novel strategies, represent a new foundation of effective therapeutic agents to breast cancer. ErbB3/EGFR/MUC1 complex formation under MUC1 expression also represents a druggable target for ErbB3 activity and stability.

Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.