Activity-based protein profiling of host-virus interactions

Abstract

Virologists have benefited from large-scale profiling methods to discover new host-virus interactions and to learn about the mechanisms of pathogenesis. One such technique, referred to as activity-based protein profiling (ABPP), uses active site-directed probes to monitor the functional state of enzymes, taking into account post-translational interactions and modifications. ABPP gives insight into the catalytic activity of enzyme families that does not necessarily correlate with protein abundance. ABPP has been used to investigate several viruses and their interactions with their hosts. Differential enzymatic activity induced by viruses has been monitored using ABPP. In this review, we present recent advances and trends involving the use of ABPP methods in understanding host-virus interactions and in identifying novel targets for diagnostic and therapeutic applications.

Figure 1

ABPP is a functional proteomics technique that uses ABPs to react covalently with the active site of mechanistically related classes of enzymes. (a) The chemical structure of an ABP consists of two essential components: a warhead reactive group (e.g. small molecule inhibitors, substrate-based scaffolds or protein-reactive molecules) that covalently targets the catalytic amino acid residue of an enzyme active site, and a reporter tag (fluorophore or biotin) for detection or purification. The linker region is a flexible chain of varying length and hydrophobicity that connects and acts as a spacer between the warhead and the bulky fluorophore tag. (b) A clickable linker most commonly exploits copper-catalyzed N₃–alkyne cycloaddition to couple a chemically inert alkyne (≡) group on the ABP to an N₃ group on the reporter tag. Click chemistry has been applied to the field of ABPP, allowing the active proteome to be labeled in situ and in vivo. (c) Examples of the three main classes of ABPs and reaction with their respective enzyme targets. Upper panel: mechanism-based ABPs are based on irreversible enzyme inhibitors, such as fluorophosphonate, and form a covalent bond with the catalytic nucleophile amino acid residue in the active site of the targeted enzyme. Depicted here is a fluorophosphonate ABP targeting a member of the serine hydrolase superfamily. Middle panel: substrate-directed ABPs depend on substrate-based scaffolds (usually an amino acid residue or a peptide, such as ubiquitin) that act as a specificity region targeting the probe to the catalytic active site of the enzyme. Following recognition of the substrate-based scaffold, the catalytic nucleophile of the enzyme (Enz^–) cleaves the scissile bond in proximity of the substrate scaffold to generate anion (X^–) that facilitates departure of the fluoride leaving group (F^–), generating a highly reactive electrophilic quinolimine methide intermediate, that reacts with a nucleophilic residue (Y^–) within the enzyme active site to bind covalently the probe to the enzyme. Lower panel: non-directed ABPs contain a mild reactive group of electrophiles, such as sulfonate ester, that have intermediate reactivity; enough to modify the catalytic nucleophile amino acids in their activated state, but low enough to prevent unspecific labeling of other nucleophile residues outside the active site. Depicted here is the sulfonate ester ABP that targets several mechanistically distinct classes of enzymes. A more detailed description of the different ABP classes, the history and discovery of warhead reactive groups and the ABP labeling mechanisms can be found elsewhere .

Figure 2

Applications of ABPP to study viral replication and infection. (a) Comparative ABPP has been extensively used to screen for differentially active enzymes during viral propagation with the potential to become therapeutic and diagnostic candidates. (b) Competitive ABPP is being used to screen inhibitor libraries and determine the specificity and sensitivity of potential therapeutics against both host and viral enzymes involved in the viral life cycle. (c) Comparative and imaging ABP probes have been used in situ by using the bioorthogonal click chemistry to monitor the variability of subcellular enzymatic activity induced by viral pathogenesis and the general experimental flow is depicted.

Figure 3

Schematic stepwise example of how the altered function of a differentially active protein identified by ABPP is linked to viral pathogenesis, such as HCV . (a) The activity profile of serine hydrolases during HCV replication was obtained with a fluorophosphonate (FP) ABP. Following gel separation and comparison of the labeled proteomes from non-infected and HCV-infected hepatocytes, a differentially labeled protein was identified by LC-MS/MS to be human CES1. (b) To determine the effect of CES1 activity on HCV propagation, the expression of CES1 was knocked down and overexpressed while monitoring the levels of HCV. As CES1 activity was found to influence the HCV life cycle, its specific role during HCV propagation was narrowed down to the metabolism of inert lipids [i.e. cholesterol esters (CEs) and triglycerides (TGs)] and their storage into intracellular LDs. (c–f) Based on the findings that CES1 favors CE and TG loading in LDs, a hypothetical synergy between CES1 and HCV has been suggested: the HCV-induced high expression of the endoplasmic reticulum (ER) protein CES1 loads TGs and CEs in LDs, increasing their size and number (d). (e) Upon LD saturation with these inert lipids, the ER–LD interstitial space increases, creating a favorable environment where high HCV replication occurs. (f) At the viral budding stage of HCV, an increase in the turnover dynamics of HCV particles allows CES1 and HCV to occupy the abundant LD–ER interface abundantly.