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ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment.

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment. [ABSTRACT FROM AUTHOR]