Vitamin E-based glycoside amphiphiles for membrane protein structural studies.

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile-lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study.

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human ß2 adrenergic receptor (ß2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of ß2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast.

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named 'ingression progression complexes' (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role.

Synchronization of the Budding Yeast Saccharomyces cerevisiae.

A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARß/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket.