It was previously shown by the Bottini Lab that PTP4A1 promotes TGFβ signaling and fibrosis in Systemic Sclerosis (SSc) via its interaction with SRC kinase (1). This thesis aims at further characterizing this interaction and looking for any factors potentially affecting it. Recombinant proteins were purified from Escherichia coli. Two in vitro co-precipitation assays were established and optimized to look at the interaction reliably and reproducibly, and the effect of various factors was examined with the assistance of the Surface Plasmon Resonance (SPR) assay. The characterization led us to propose an interaction mechanism between PTP4A1 and SRC which results in the enhancement of the pro-fibrotic signaling triggered by TGFβ. The relevance of the interaction was also confirmed in SSc dermal fibroblasts and mouse tissue using Proximity Ligation Assay (PLA) performed by other Bottini lab members. Together, these data further demonstrate the critical role of the PTP4A1/SRC interaction in SSc and indicate that PTP4A1 might serve as a therapeutic target.

Protein Tyrosine Phosphatases (PTPs) are drug target candidates due to their role in signal transduction and involvement in various pathologies. Difficulty in developing orthosteric inhibitors of PTPs suitable for therapeutic purposes has raised interest in the development of allosteric inhibitors, yet no allosteric regulatory model has been formulated for most PTP subtypes. For the two PTPs, belonging to two distinct subtypes, for which such mechanisms have been characterized, SHP2 and PTP1B, they have paved the way to development of specific inhibitors which show promise for future development of small molecule therapeutics. In this study we achieved the formulation and in vitro validation of a novel structure-based allosteric model for RPTPα and discuss its mechanistic implications, potential for drug discovery and possible applicability to other PTPs.

T-cell protein tyrosine phosphatase (TCPTP) is a classical non-transmembrane PTP that is expressed in every cell, despite its name. In its catalytic domain, it shares 65% similarity identity to PTP1B, which was the first tyrosine phosphatase to be identified, and the most thoroughly researched. Based on its similarity with PTP1B, TCPTP is predicted to have a 280 amino acid catalytic domain followed by the regulatory α helix 7 (α7), and a C-terminal intrinsically disordered region. Allosteric inhibitors that target α7 and the C-terminal tail of PTP1B have been characterized. Since the active sites of classical PTPs have generally similar features, targeting the active site leads to inhibitors with poor specificity. Therefore, domains that regulate the PTP catalytic activity have been the focus of much recent research. In this study, we will be investigating the role of the C-terminal domain of TCPTP. The regulation of TCPTP by the C-terminal domain are not clearly defined. By knocking out TCPTP and reconstituting the wild-type full length and catalytic domain, we will assess the effect of the C-terminal domain on TCPTP activity in a cellular context. By combining mutagenesis, enzymatic assays, and FRET assays, we will investigate the areas in the protein and mechanisms that are responsible for its inhibition and activation in cells and in vitro.

Rheumatoid arthritis is a chronic, progressive autoimmune disease in which fibroblast like synoviocytes (FLS) play a key role. In the pathogenesis of the disease, FLS infiltrate the joints and promote inflammation and destruction of joint cartilage. Tranmembrane protein tyrosine phosphatase receptor type alpha (PTPRA), a known activator of the kinase Src, is highly expressed in RA FLS and promotes their aggressiveness. Knockout of PTPRA protects against joint swelling and disease progression in KBxN models of mice arthritis. A canonical model of transmembrane PTP regulation is that they are inactivated by dimerization. However, these models were never validated by single cell assessments of full length PTPs in primary cells. Here, through FRET microscopy and functional assays of multiple independent primary PTPRA knockout murine FLS cell lines transfected with PTPRA mutant constructs, we show that PTPRA dimerization occurs in FLS and positively correlates with PTPRA association with Src and promotion of cell motility. PTPRA mutants impairing dimerization of PTPRA and its association to Src at the leading edge of migrating mouse FLS also displayed impaired cell morphology and motility. These results are apparently inconsistent with the aforementioned canonical model of transmembrane PTP regulation, indicating the need for further investigations of PTPRA regulation.

Chronic autoimmune disorders collectively affect 5-7% of the global population and are a major public health concern. The prevailing paradigm for autoimmune disease treatment relies on immunosuppression, which can be effective but leaves patients susceptible to opportunistic or serious infections and cancer. Moreover, these therapies are not designed to correct immune dysfunction that underlies autoimmunity. For those that continue to experience disease symptoms, there is an unmet need for therapies that operate via immunoregulation and avoid generalized immunosuppression. A key challenge is that unlike diseases with known etiology, the pathogenesis of autoimmune diseases can be complex. However, a common feature involves hyperactivated immune cells that, left unchecked, can lead to permanent damage of healthy tissue. To this end, a key defect arises from a loss in the number and function of autoimmune-protective cells called regulatory T cells (Treg) that normally prevent immune responses against one’s own cells and tissues. The premise of this dissertation is that enhancing Treg can be harnessed to promote disease-specific immunoregulation without causing generalized immunosuppression. To test the premise, the work reported herein describes the development and applications of biomaterial-based disease modifying agents. Three methods are described. The first method described used an immunomodulatory nanocomplex formulation that differentially modulated immune cell metabolism to enhance Treg over inflammatory T cells. A kinetic model describing Treg enhancement confirmed a strong dependence on the initial T cell population. The second and third methods described demonstrated that the epigenetic modulation of immune cells can strongly influenced the immunophenotypic trajectories of T cells that favor a Treg phenotype. Further, the formulation of a novel sustained biomaterial designed to locally enhance Treg via epigenetic modulation and its application in a model of inflammatory arthritis is reported. Overall, this work paves the way for the systematic design and validation of immunoregulatory biomaterials for the treatment of autoimmune disorders.

Protein Tyrosine Phosphatases (PTPs) are drug target candidates due to their role in cell signaling and involvement in the pathologies of various diseases. Difficulty in developing orthosteric inhibitors of PTPs has raised interest in the development of allosteric inhibitors. Previous studies have identified an allosteric mechanism in receptor-type protein tyrosine phosphatase α (RPTPα), yet the mechanism requires further characterization for future development of small molecule therapeutics. In this study, through comparing RPTPα and the closely related RPTPε, we achieved the identification and in vitro validation of a segment of RPTPα (residues 261-330) responsible for its allosteric effect. It lays the groundwork for identification of amino acid residues participating in the establishment of allostery, which is a step in fully elucidating the allosteric mechanism.

Fibrosis is a condition characterized by tissue overgrowth, hardening, and /or scarring. It is typically caused by excess deposition of extracellular matrix (ECM) components. Fibroblasts play an important role in the maintenance and reabsorption of the ECM, and thus can be critical mediators of the condition. Protein Tyrosine Phosphatase Receptor Type Gamma (PTPRG) is highly expressed in fibroblasts, and so we want to delve into how PTPRG activation and inactivation may play a role in the condition. The phosphatase activity of receptor-type protein tyrosine phosphatases (RPTPs) is widely thought to be regulated through dimerization, however, the dimerization of PTPRG in fibroblast activity has not been seen in primary cells. Thus, we used FRET microscopy on a primary PTPRG knockout murine dermal fibroblast cell line (mDF) transfected with PTPRG mutant constructs to observe full-length protein dimerization in a cellular context. We also performed biochemical observations of protein activity through phosphatase activity experiments. We found that WT and several mutants of PTPRG dimerize in mDFs. A dimerization-inactivating mutant exhibits less dimerization in cells and in vitro.