MEASURING THE THERMODYNAMICS OF RECEPTOR TYROSINE KINASE INTERACTIONS: A STUDY OF HOMODIMERS, HETERODIMERS, AND RECEPTOR-ADAPTER PROTEIN COMPLEXES

Protein-protein interactions regulate many biological systems. Understanding these interactions can provide insight into the function of proteins and the design of biological signaling networks, and can inform the development of more effective therapeutics. Protein-protein interactions can be described by thermodynamics, that is, the strength of binding, as well as by the distance between binding partners. In this thesis, I quantify protein-protein interactions in the Receptor Tyrosine Kinase (RTK) family, using novel quantitative fluorescence microscopy methods. In Chapter Three and Chapter Four, I characterize the formation of RTK homodimers using existing techniques. Specifically, I examine a subfamily of RTKs, known as the Fibroblast Growth Factor Receptors (FGFRs), in the presence of (i) pathogenic point mutations and (ii) activating ligands. In Chapter Five, I consider the formation of RTK heterodimers. I develop a method of measuring the thermodynamics of RTK heterodimer formation and apply it to the FGFR family. I also examine the effects of pathogenic point mutations on FGFR heterodimers. In Chapter Six, I study the interaction of RTKs with adapter proteins, an event that directly links RTKs to intracellular signaling networks. I develop and implement a procedure to measure the thermodynamics of this binding reaction, using the RTK Epidermal Growth Factor Receptor (EGFR) and the adapter protein Growth factor receptor-bound protein 2 (Grb2) as a model system. Overall, the work in this thesis demonstrates the utility of fluorescence microscopy for the quantitative characterization of protein-protein interactions in the membrane and reports previously unknown properties of the protein-protein interactions that regulate the RTK activation process

Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

AbstractHere, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova

Tumor-on-chip modeling of organ-specific cancer and metastasis

Every year, cancer claims millions of lives around the globe. Unfortunately, model systems that accurately mimic human oncology - a requirement for the development of more effective therapies for these patients - remain elusive. Tumor development is an organ-specific process that involves modification of existing tissue features, recruitment of other cell types, and eventual metastasis to distant organs. Recently, tissue engineered microfluidic devices have emerged as a powerful in vitro tool to model human physiology and pathology with organ-specificity. These organ-on-chip platforms consist of cells cultured in 3D hydrogels and offer precise control over geometry, biological components, and physiochemical properties. Here, we review progress towards organ-specific microfluidic models of the primary and metastatic tumor microenvironments. Despite the field\u27s infancy, these tumor-on-chip models have enabled discoveries about cancer immunobiology and response to therapy. Future work should focus on the development of autologous or multi-organ systems and inclusion of the immune system

Advances in modeling the immune microenvironment of colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death in the US. CRC frequently metastasizes to the liver and these patients have a particularly poor prognosis. The infiltration of immune cells into CRC tumors and liver metastases accurately predicts disease progression and patient survival. Despite the evident influence of immune cells in the CRC tumor microenvironment (TME), efforts to identify immunotherapies for CRC patients have been limited. Here, we argue that preclinical model systems that recapitulate key features of the tumor microenvironment-including tumor, stromal, and immune cells; the extracellular matrix; and the vasculature-are crucial for studies of immunity in the CRC TME and the utility of immunotherapies for CRC patients. We briefly review the discoveries, advantages, and disadvantages of curren

MEASURING THE THERMODYNAMICS OF RECEPTOR TYROSINE KINASE INTERACTIONS: A STUDY OF HOMODIMERS, HETERODIMERS, AND RECEPTOR-ADAPTER PROTEIN COMPLEXES

Protein-protein interactions regulate many biological systems. Understanding these interactions can provide insight into the function of proteins and the design of biological signaling networks, and can inform the development of more effective therapeutics. Protein-protein interactions can be described by thermodynamics, that is, the strength of binding, as well as by the distance between binding partners. In this thesis, I quantify protein-protein interactions in the Receptor Tyrosine Kinase (RTK) family, using novel quantitative fluorescence microscopy methods. In Chapter Three and Chapter Four, I characterize the formation of RTK homodimers using existing techniques. Specifically, I examine a subfamily of RTKs, known as the Fibroblast Growth Factor Receptors (FGFRs), in the presence of (i) pathogenic point mutations and (ii) activating ligands. In Chapter Five, I consider the formation of RTK heterodimers. I develop a method of measuring the thermodynamics of RTK heterodimer formation and apply it to the FGFR family. I also examine the effects of pathogenic point mutations on FGFR heterodimers. In Chapter Six, I study the interaction of RTKs with adapter proteins, an event that directly links RTKs to intracellular signaling networks. I develop and implement a procedure to measure the thermodynamics of this binding reaction, using the RTK Epidermal Growth Factor Receptor (EGFR) and the adapter protein Growth factor receptor-bound protein 2 (Grb2) as a model system. Overall, the work in this thesis demonstrates the utility of fluorescence microscopy for the quantitative characterization of protein-protein interactions in the membrane and reports previously unknown properties of the protein-protein interactions that regulate the RTK activation process

Microneedle-mediated nanomedicine to enhance therapeutic and diagnostic efficacy

Nanomedicine has been extensively explored for therapeutic and diagnostic applications in recent years, owing to its numerous advantages such as controlled release, targeted delivery, and efficient protection of encapsulated agents. Integration of microneedle technologies with nanomedicine has the potential to address current limitations in nanomedicine for drug delivery including relatively low therapeutic efficacy and poor patient compliance and enable theragnostic uses. In this Review, we first summarize representative types of nanomedicine and describe their broad applications. We then outline the current challenges faced by nanomedicine, with a focus on issues related to physical barriers, biological barriers, and patient compliance. Next, we provide an overview of microneedle systems, including their definition, manufacturing strategies, drug release mechanisms, and current advantages and challenges. We also discuss the use of microneedle-mediated nanomedicine systems for therapeutic and diagnostic applications. Finally, we provide a perspective on the current status and future prospects for microneedle-mediated nanomedicine for biomedical applications.</p

Production of Plasma Membrane Vesicles with Chloride Salts and Their Utility as a Cell Membrane Mimetic for Biophysical Characterization of Membrane Protein Interactions

Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT. We characterize the size distribution of the produced vesicles. We also show that these vesicles can be used for the biophysical characterization of interactions between membrane proteins

Advances in Modeling the Immune Microenvironment of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death in the US. CRC frequently metastasizes to the liver and these patients have a particularly poor prognosis. The infiltration of immune cells into CRC tumors and liver metastases accurately predicts disease progression and patient survival. Despite the evident influence of immune cells in the CRC tumor microenvironment (TME), efforts to identify immunotherapies for CRC patients have been limited. Here, we argue that preclinical model systems that recapitulate key features of the tumor microenvironment-including tumor, stromal, and immune cells; the extracellular matrix; and the vasculature-are crucial for studies of immunity in the CRC TME and the utility of immunotherapies for CRC patients. We briefly review the discoveries, advantages, and disadvantages of current in vitro and in vivo model systems, including 2D cell culture models, 3D culture systems, murine models, and organ-on-a-chip technologies