INSIGHTS INTO THE STRUCTURE, FUNCTION, AND INHIBITION OF SHIP1: A POTENTIAL THERAPEUTIC TARGET FOR THE TREATMENT OF LATE-ONSET ALZHEIMER’S DISEASE (LOAD)
Phosphatidylinositol phosphates (PIPs) and soluble inositol phosphates (IPs) serve as critical secondary messenger molecules that regulate cellular processes. The INPP5 family of phosphatases play an essential role in regulating levels of PIP-5’ and IP-5’ molecules. Src homology 2-containing-inositol phosphatases (SHIP), are a subgroup of the INPP5 family that consists of two members, SHIP1 and SHIP2. Both SHIP proteins have been identified to hydrolyze PI(3,4,5)P3 into PI(3,4)P2. Interestingly, the dysregulation of PI(3,4,5)P3 and SHIP proteins have been observed in multiple diseases, such as cancer, diabetes, and neurodegenerative disease. Recently, SHIP1 was identified as a potential risk factor for the development of Late-onset Alzheimer’s Disease (LOAD). Furthermore, knockdown and inhibition of SHIP1 using small-molecule inhibitors were shown to reduce phenotypes associated with LOAD. Taking these studies together suggests SHIP1 to be a potential therapeutic target for the treatment of LOAD.
Despite SHIP1’s therapeutic potential, the development of specific small-molecule inhibitors that target SHIP1 has been challenging. One explanation for this challenge is that very little is known about the overall structure and function of SHIP1. In this thesis I will discuss in detail how we generated multiple SHIP1 constructs to improve our understanding of SHIP1’s overall structure and function in an in vitro setting.
Efficient protein production is essential for studying enzyme structure and function. The choice of expression system can impact protein yield and stability. The E. coli (BL21) and Baculovirus expression systems are two commonly used systems for protein production. While E. coli is cost-effective and can yield a large amount of protein, the Baculovirus system offers advantages in terms of protein folding and post-translational modifications. Using both systems to generate SHIP1 protein, we demonstrate that the Baculovirus system significantly enhances SHIP1 solubility for all generated constructs, making it the preferable choice for investigating the structure and function of SHIP1.
SHIP1, a 133 kDa protein, which comprises five established domains: an N-terminal Src Homolgy 2 (SH2) domain, 2.) a pleckstrin homology-related (PH) domain, 3.) an inositol phosphatase catalytic (Ptase) domain, 4.) a C2 domain, and 5.) a C-terminal domain containing proline-rich regions (PXXP) and tyrosine phosphorylated (NPXY) motifs. Despite their regulatory roles in phosphatase activity, protein-protein interactions, and membrane association, limited information is available about their structures and how they contribute SHIP1’s biochemical functions. In this study, we utilized baculovirus-expressed SHIP1 constructs to investigate the impact of each domain on macromolecular structure. Interestingly, a previously unrecognized domain within SHIP1 that directly impacts the enzyme's oligomeric state was identified. This work highlights that SHIP1's individual domains can significantly impact its overall structure and function, providing valuable insights for the development of potential therapeutics in the treatment of LOAD.
Accurate determination of phosphatase kinetics is vital for understanding the enzymatic activity and its potential involvement in disease. Using our baculovirus generated SHIP1 constructs, we employed in-vitro assays, including the malachite green (MG) and the 2-amino-6-mercapto-7-methylpurine riboside (MESG) coupled enzyme assays, to gain insight into SHIP1 kinetics. Results from the MG assay shows that SHIP1 can hydrolyze the PI(3,4,5)P3 diC8 substrate more efficiently than I(1,3,4,5)P4. Additionally, SHIP1’s PH domain was observed to increase the turnover of PI(3,4,5)P3 diC8. Furthermore, dimerization of SHIP1 was not observed to alter SHIP1 kinetics in any way. Lastly, no major differences in I(1,3,4,5)P4 kinetics were observed with the addition of SHIP1’s N-terminus. These results offer the first comprehensive biochemical characterization of SHIP1 across its substrates and N-terminal domains.
The development of potent and specific small-molecule inhibitors that target SHIP1 remains challenging. One potential cause for this challenge is that no structures of SHIP1 have been solved in complex with active compounds, making structure-based drug design impossible. In this study, we developed a covalent compound, TAD-58547, from a previously published fragment-based screen that was conducted on SHIP1’s Ptase and C2 domain. TAD-58547 was shown to effectively inhibited SHIP1's Ptase and C2 domains at modest potency. Using X-ray crystallography, this compound was observed to form a covalent interaction with a cysteine residue near the Phosphatase-C2 domain interface. Intriguingly, the inhibitor's potency was observed to be reduced in the presence of the SH2 domain. In addition to testing TAD-58547 against our SHIP1 constructs, we investigated the effect of SHIP1’s N-terminus on the potency of a literature compound, TAD-58616. This compound was shown to inhibit all our tested constructs at low µM concentrations. Furthermore, using x-ray crystallography TAD-58616 was solved in complex with SHIP1’s Ptase and C2 domain. Intriguingly, density for TAD-58616 was shown to interact with a site previously identified from the fragment-based screen. While we initially determined this site to be a result of crystal packing, fragments bound to this site may have the potential to inhibit SHIP1. The work presented in this study reinforced the importance of testing inhibitors against physiological relevant forms of SHIP1, when developing potential therapeutics.
Lastly, new evidence has suggested that the binding of phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) and immunoreceptor tyrosine-based inhibitory motifs (p-ITIM) to SHIP1’s N-terminal SH2 domain is essential for its “Anchorage and Activation” at the plasma membrane (PM). With this model it is believed that SHIP1’s SH2 domain, places the phosphatase into an auto-inhibited state. Upon binding to immune receptor proteins and adaptor proteins that contain ITAM/ITIM sequences, SHIP1 becomes un-auto-inhibited, allowing it to efficiently hydrolyze PI(3,4,5)P3 embedded in the PM. While this model does support the notion that SHIP1 activity is mediated by its PM localization, our biophysical and biochemical characterization add another level of complexity to this regulatory event. Taking all these results together, we propose a novel model for SHIP1 called “Anchorage and Assist” and suggest innovative therapeutic strategies for targeting SHIP1.
In conclusion, this thesis highlights the importance of choosing suitable expression systems for efficient protein production. Additionally, it offers insight into SHIP1's regulatory mechanisms through the discovery of a novel domain impacting its oligomeric state. Furthermore, the accurate determination of SHIP1 kinetics enhances our understanding of this phosphatase and its potential implications in disease. Also, the identification and crystallization of a novel and previously determined inhibitor scaffolds in complex with SHIP1 increases our ongoing efforts to develop a small-molecule inhibitor that specifically targets SHIP1. Lastly, using recently published data, detailing SHIP1 PM localization and activation, we proposed a new model for SHIP1 activity and suggest novel therapeutic strategies for targeting SHIP1.
- Doctor of Philosophy
- West Lafayette