December 2011

New protein sensors to quantify phosphoinositides in situ

 

Lipid_News_Fig1 
Figure 1. The epsin1 ENTH domain (PDB 1HOA) was used to engineer a high-affinity PI(4,5)P2 binding reporter. Methionine 10 (magenta) was mutated to cysteine to attach the environmentally sensitive probe, while serine 4 (green) was mutated to tryptophan to increase membrane affinity. Inositol(1,4,5)P3 bound to the ENTH domain is shown in red.

Cellular membranes harbor receptors, ion channels, lipid domains, lipid signals and scaffolding complexes that function to maintain cellular growth, metabolism and homeostasis (1). Abnormalities in lipid metabolism attributed to genetic changes, among other causes, are associated with a host of diseases (2). Thus, there is a need to understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development.

The lipid bilayer has a highly polarized structure that consists of a central hydrocarbon core and two flanking interfacial regions that are highly dynamic and could contain thousands of different lipids (1). This dynamic variety of glycerolipids, sphingolipids and sterols in membrane organelles provide spatial and temporal cues to direct signaling processes through target proteins (3). However, there remains a large gap in our understanding of the spatial and temporal dynamics of the lipids that produce these bioactive signals.

Given that nearly half of all proteins are located in or on membranes, it is not surprising that there are a variety of conserved lipid-binding domains in eukaryotes. Some of these domain families rank in the top 15 modular domains in the human genome and are most often found in signal-transduction and membrane-trafficking proteins (4). To date, fluorescently tagged lipid-binding domains (such as the PH domain) that harbor high specificity and affinity for phosphoinositides (PIs) have most often served to study PI dynamics and localization (5). While the overall spatial distribution of lipids such as PI(3)P and PI(4,5)P2 (5) is well appreciated, the actual concentration, distribution and spatiotemporal dynamics have not been determined quantitatively. Thus, real-time lipid sensors that could provide high sensitivity for a specific PI to quantify its role in a cellular-signaling cascade would be a great advantage to researchers.

Recently, Wonhwa Cho and colleagues developed such an approach to quantify PI(4,5)P2 using a chemically modified lipid-binding domain (6).

The probe was first engineered for optimal lipid-binding properties and minimal affinity for cellular proteins. Through the introduction of an environmentally sensitive chemical probe on a free cysteine, the engineered domain serves as a turn-on sensor that undergoes a large increase in fluorescence upon lipid binding.

 

 

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