Activity-Based Phosphatidylinositol Kinase Probes Detect Changes to Protein–Protein Interactions During Hepatitis C Virus Replication

Abstract

Protein-protein interactions are integral to host-virus interactions and can contribute significantly to enzyme regulation by changing the localization of both host and viral enzymes within the cell, inducing conformational change relevant to enzyme activity or recruiting other additional proteins to form functional complexes. Identifying the interactors of active enzymes using an activity-based protein profiling probe has allowed us to characterize both normal enzyme activation mechanisms and the manner by which these mechanisms are hijacked and altered by the hepatitis C virus (HCV). Here, we report use of a novel activity-based probe, PIKBPyne, which labels phosphatidylinositol kinases (PIKs) in an activity-based manner, to investigate HCV-dependent changes in protein-protein interactions for PI4KB. Herein, we report the synthesis of new variations on PIKBPyne, compare their ability to label the interacting partners of PI4KB, and demonstrate the utility of our approach in characterizing virus-mediated changes to host function.

ABPP and Host–Virus Interactions

“…These flexible-linker PIKBPyne probes were shown to be able to label proteins interacting with active PI4KB. Labelling with PIKBPyne demonstrated an HCV-induced decrease in the interaction between PI4KB and the Golgi-resident protein ACBD3 (Desrochers et al 2018). It had previously been reported that HCV induced a change in the localisation of PI4KB from the Golgi towards sites of viral replication (Zhang et al 2012).…”

“…PI4KB activity is known to be regulated by stable protein-protein interactions which recruit PI4KB to membranes and stabilise an activating phosphorylation at Ser294 (Hausser et al 2005(Hausser et al , 2006Balla 2013). To investigate the role of these interactions in the post-translational regulation of PI4KB activity, Desrochers et al synthesised new PIKBPyne probes containing flexible linkers of varying length between the targeting PIK93 moiety and the crosslinking benzophenone (Desrochers et al 2018). These flexible-linker PIKBPyne probes were shown to be able to label proteins interacting with active PI4KB.…”

Bifunctional Molecular Probes for Activity‐Based Visualization of Quinone‐Dependent Amine Oxidases

“…Pioneering work in this field by the Cravatt group focused on using fluorophosphonate‐containing bifunctional molecular probes to visualize the family of serine protease enzymes . These researchers and others have continued to expand the field, for example, by developing molecular probes to report various other families of enzymes, by using probes to identify proteins whose active forms are up‐regulated in disease states, such as in metastatic versus benign cancers, by using these probes to visualize how inhibitors affect whole families of enzymes within full proteome mixtures to inform inhibitor discovery, and by combining with photoaffinity strategies to identify protein–protein interactions . Similar molecular probes and strategies have also been used to study families of post‐transitionally modified non‐enzymatic proteins.…”

A Photoaffinity Displacement Assay and Probes to Study the Cyclin‐Dependent Kinase Family

“…[5,6] Thed esign of the probe,i ncluding choice of photoreactive group,p rofoundly impacts the likelihood of successful protein capture.T here have been few studies to systematically compare the efficiency of crosslinking of commonly used photoreactive groups and there is still no consensus on the ideal photoreactive group and attachment strategy. [7][8][9] Biochemical studies into the determinants of crosslinking yields will help inform on the design of photoaffinity probes for applications of interest.…”