PTP1B: Studies of its substrate specificity and an investigation into rational drug design

Abstract

The regulation of cellular signaling processes is paramount for the maintenance of cellular homeostasis. Phosphorylation is one component of the signaling apparatus utilized by cells for the regulation of such cellular processes as growth, neuronal development, the cell cycle, and metabolism (Zhang, 1998; Neel & Tonks, 1997). Because of the importance and ubiquitous nature of phosphorylation in cellular processes, it is not surprising that the genome of higher eukaryotes contains approximately 2000 protein kinase and 1000 phosphatase genes (Hunter, 1995). In order to decipher the cellular language of signal transduction and to confront disease states in which disruption of these cellular signaling mechanisms is causative, the study of enzymes that catalyze the addition of phosphate and its removal must be advanced.;The protein phosphatase (PP) superfamily is composed of three distinct gene families. Comprising the Ser/Thr phosphatases are two enzyme families, which dephosphorylate phosphoserine and phosphothreonine residues, the PPP and PPM enzymes. The third family, the protein tyrosine phosphatases (PTPs), composed of the tyrosine specific and dual-specificity phosphatases, dephosphorylate phosphotyrosine as well as phosphoserine and phosphothreonine residues, respectively. The focus of this work is the tyrosine phosphatase PTP1B. PTP1B was the first discovered cytoplasmic PTP and represents the prototypic PTP. First found in human placenta, PTP1B is now known to be ubiquitously expressed and implicated in numerous cellular signaling pathways, such as cancer and diabetes. Not surprisingly, the involvement of PTP1B in various pathologies has made this enzyme a target of much study.;My work sought to address questions regarding PTP1B substrate specificity. Such as, what are the structural features which are involved in substrate recognition and how they function to bind substrate, and is whether PTP1B an enzyme with defined substrate specificity or instead is promiscuous. In order to tackle this issue, I first employed site-directed mutagenesis coupled with mechanistic enzymology. In addition to revealing novel functions for specific residues in the catalytic mechanism, I was able to demonstrate that PTP1B does indeed house inherent substrate specificity. However, it also possesses additional innate structural features that impart a measure of plasticity and it is this plasticity that grants PTP1B the ability to recognize peptide substrates of highly variable sequence. Since the structural features which grant PTP1B this plasticity were previously entirely unknown, I next decided to employ protein crystallography to reveal how this enzyme's recognition apparatus functions. Together with another crystal structure of PTP1B bound with a peptide substrate, the structure of PTP1B bound with a peptide substrate has unveiled the structural features of PTP1B plasticity and has indeed enhanced our understanding of PTP1B substrate specificity. Finally, incorporating our gained knowledge of PTP1B specificity, I decided to also explore the realm of PTP1B inhibitors. Since the availability and development of potent PTP1B inhibitors is limited, I decided to employ novel computational techniques for the purpose of identifying new PTP1B inhibitors. This effort has led to the discovery of novel phosphate mimics and a new structural class of PTP1B inhibitors. Potent PTP1B inhibitors can be of great use in determining the in vivo specificity of the enzyme and can potentially be used therapeutically.