Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Singh, Nilmani

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https://hdl.handle.net/2142/109506 Copy

Description

Title

Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Author(s)

Singh, Nilmani

Issue Date

2020-11-30

Director of Research (if dissertation) or Advisor (if thesis)

Chen, Jie

Doctoral Committee Chair(s)

Chen, Jie

Committee Member(s)

• Prasanth, Supriya
• Freeman, Brian
• Fratti, Rutilio

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• PH domain
• Human PH domain proteins
• SiMPull
• Single molecule pull down
• TIRF microscopy
• Lipid-Protein Interactions
• ARHGEF3
• XPLN
• RhoGEF
• AKT
• lipid binding prediction
• probabilistic sequence comparison
• RFC
• Pleckstrin Homology domain
• PIP
• phosphatidylinositol

Abstract

Lipids and proteins are two major components of cells, which are crucial for structural integrity and maintenance of cellular homeostasis by integrating various extracellular and intracellular cues. Lipids are involved in the regulation of cellular functions and can interact with proteins and undergo diverse chemical modifications. The presence of distinct lipid species creates a unique membrane signature for various cellular organelles as well as acts as a localization signal for multiple proteins. Proteins have evolved to contain specific lipid-binding modules crucial for mediating protein-lipid interaction. Numerous human pathologies have been linked to perturbations in lipid signaling and lipid-protein interactions, thus underscoring the need to study protein-lipid interactions and associated cellular processes. In this dissertation, I have examined the binding characteristics of Pleckstrin Homology (PH) domain, a lipid-binding domain, with phosphoinositides (phosphatidylinositol phosphates or PIPs), an important class of regulatory phospholipids. PH domains are presumed to bind PIPs, but specific interaction with and regulation by PIPs for most PH domain-containing protein remains unclear. Recapitulating biologically relevant protein-lipid interactions in vitro is challenging due to a myriad of membrane properties such as curvature, composition, membrane asymmetry, viscosity, lipid rafts etc. The traditional methods to investigate these interactions rely on purified proteins, isolated lipid-binding domains, or non-physiological lipid environments. These common techniques include lipid strips, liposome sedimentation, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR); however, despite their wide use, they are known to be artifact-prone because of their reliance on purified proteins, isolated domains, and/or non-physiological lipid presentation. To overcome those shortcomings, our laboratory has developed a single-molecule pull down (SiMPull) assay to probe protein-lipid binding. This method, called lipid-SiMPull, has high specificity and sensitivity while utilizing lipid vesicles of desired composition and full-length proteins expressed in whole cell lysates without the need for purification. In Chapter 2, utilizing lipid-SiMPull, I have examined the PIP binding for 67 full-length human PH domain-containing proteins. Approximately half of them were found to have affinity for PIPs with various specificity, the majority of which had not been previously reported. For 8 proteins, PI(3,4,5)P3 (PIP3) binding affinity was further validated in vivo by insulin stimulated PIP3 production on the plasma membrane and subsequent translocation. In Chapter 3, I describe a probabilistic sequence comparison algorithm, called recursive functional classification (RFC), created using the lipid-SiMPull data that identified the amino acid determinants of PIP binding throughout the entire sequence of the PH domain. The results from lipid-SiMPull experiments and RFC algorithm suggest that about half of the 246 human PH domains should bind PIPs with some specificity. Guanine nucleotide-exchange factors (GEFs) catalyze the GTP-bound active state of small G proteins. The Rho family of small G proteins are activated by a large family of proteins containing Dbl-homology (DH) domain, called RhoGEFs, which are known for conserved DH-PH domain structures. While the DH domain is responsible for GEF activity, the function of the PH domain in RhoGEFs is not well-understood. In Chapter 4, I have investigated the role of phosphoinositide binding by ARHGEF3, a RhoGEF, in its biological functions. In the lipid-SiMPull screening, ARHGEF3 was found to specifically bind PI(4,5)P2 and PI(3,5)P2 in Lipid-SiMPull screening through its PH domain. Of the two known biological function of ARHGEF3, lipid-binding regulates the GEF activity towards RhoA and but not the inhibition of mTORC2 kinase. This adds to the growing understanding of the structure-function relationship for RhoGEFs, especially the poorly-understood role of the conserved PH domains. AKT is a serine/threonine kinase crucial for regulation of diverse cellular processes and a major drug target for various human pathologies. Binding of the AKT PH domain with PIP3 or PI(3,4)P2 is responsible for its membrane translocation and activation. Different models have been proposed to explain how lipid binding modulates the activity of AKT in cells. While the classical view holds that AKT dissociates from the membrane after activation, recent studies have found that membrane dissociation results in rapid dephosphorylation and deactivation of AKT. A more detailed understanding of the lipid-binding and associated intra-molecular conformational changes is crucial to describe the activation model of AKT. In Chapter 5, I have combined lipid-SiMPull and mutagenesis to address questions regarding the role of lipid-binding in the mechanism of AKT activation. The results suggest that the hydrophobic motif domain of AKT may play an important role in inhibiting lipid-binding and that future investigation is warranted to elucidate the interactions of hydrophobic motif domain with other domains in AKT.

Graduation Semester

2020-12

Type of Resource

Thesis

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Copyright and License Information

Copyright 2020 Nilmani Singh

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