Unraveling protein dynamics to understand the brain - the next molecular frontier.

The technological revolution to measure global gene expression at the single-cell level is currently transforming our knowledge of the brain and neurological diseases, leading from a basic understanding of genetic regulators and risk factors to one of more complex gene interactions and biological pathways. Looking ahead, our next challenge will be the reliable measurement and understanding of proteins. We describe in this review how to apply new, powerful methods of protein labeling, tracking, and detection. Recent developments of these methods now enable researchers to uncover protein mechanisms in vivo that may previously have only been hypothesized. These methods are also useful for discovering new biology because how proteins regulate systemic interactions is not well understood in most cases, such as how they travel through the bloodstream to distal targets or cross the blood-brain barrier. Genetic sequencing of DNA and RNA have enabled many great discoveries in the past 20 years, and now, the protein methods described here are creating a more complete picture of how cells to whole organisms function. It is likely that these developments will generate another transformation in biomedical research and our understanding of the brain and will ultimately allow for patient-specific medicine on a protein level.

Lipid-based and protein-based interactions synergize transmembrane signaling stimulated by antigen clustering of IgE receptors.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FcεRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FcεRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FcεRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FcεRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FcεRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FcεRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.

The Role of Alcohol Dehydrogenase in Drug Metabolism: Beyond Ethanol Oxidation.

Alcohol dehydrogenases (ADHs) are most known for their roles in oxidation and elimination of ethanol. Although less known, ADHs also play a critical role in the metabolism of a number of drugs and metabolites that contain alcohol functional groups, such as abacavir (HIV/AIDS), hydroxyzine (antihistamine), and ethambutol (antituberculosis). ADHs consist of 7 gene family numbers and several genetic polymorphic forms. ADHs are cytosolic enzymes that are most abundantly found in the liver, although also present in other tissues including gastrointestinal tract and adipose. Marked species differences exist for ADHs including genes, proteins, enzymatic activity, and tissue distribution. The active site of ADHs is relatively small and cylindrical in shape. This results in somewhat narrow substrate specificity. Secondary alcohols are generally poor substrates for ADHs. In vitro-in vivo correlations for ADHs have not been established, partly due to insufficient clinical data. Fomepizole (4-methylpyrazole) is a nonspecific ADH inhibitor currently being used as an antidote for the treatment of methanol and ethylene glycol poisoning. Fomepizole also has the potential to treat intoxication of other substances of abuse by inhibiting ADHs to prevent formation of toxic metabolites. ADHs are inducible through farnesoid X receptor (FXR) and other transcription factors. Drug-drug interactions have been observed in the clinic for ADHs between ethanol and therapeutic drugs, and between fomepizole and ADH substrates. Future research in this area will provide additional insights about this class of complex, yet fascinating enzymes.

The role of carbonyl reductase 1 in drug discovery and development.

INTRODUCTION: Carbonyl reductase 1 (CBR1) plays a critical role in drug metabolism of ketones and aldehydes. CBR1 has broad substrate specificity and is involved in metabolizing a number of clinically important drugs. Areas covered: The impact of CBR1 in drug metabolism and disposition are discussed. The CBR1 enzyme is covered in detail including discussion on topics such as tissue distribution, species difference, individual variability, the effect of genetic polymorphism and disease state, iGnducibility and drug-drug interaction potential. The structure and function of CBR1 and CBR3 are also compared. In addition, the formation of chiral alcohols from CBR1 reduction and MIST coverage are reviewed. Expert Opinion: As CBR1 is an emerging enzyme in drug discovery and development, much research is needed to further understand its role in drug metabolism and disposition. In vitro-in vivo correlation for CBR1-mediated clearance is mostly unknown. Selective CBR1 inhibitors and substrates are not well enough characterized for reaction phenotyping of the CBR1 pathway. Multiple pathways appear to be involved in the regulation of CBR1. Future investigation will also help reveal their impact on drug-drug interaction potentials and the influence of individual variability.