Current Protein and Peptide Science - Volume 11, Issue 2, 2010

α1-antitrypsin (α1-AT) is a 52 kDa sialoglycoprotein. The function of α1-antitrypsin is to protect the lower respiratory tract of lungs from proteolytic degradation by neutrophil elastase. Severe genetic deficiency of α1-AT is associated with early onset emphysema and liver diseases. α1-AT also exhibits anti-inflammatory activities independent of its protease inhibitor function. There are over 90 genetic variants of human α1-antitrypsin. These variants occur due to amino acid substitution / deletion which results in charge differences. Based on charge differences these variants have been identified by isoelectric focusing. The two most common deficiency variants are S and Z. The S variant migrates anodal to Z variant. The Z variant migrates most cathodal in isoelectric focusing, hence named Z. In Z variant, the β-sheet A undergoes expansion , therefore it can easily accept the reactive site loop of a second α1-AT molecule and consequently form polymers of α1-AT. These polymers of α1-AT aggregate in the hepatocytes and show liver and lungs diseases. Contrary to this, the S variant of α1-AT is not associated with any significant clinical disease because the conformation of the inhibitor is not altered significantly. The Z related pathologies could be treated by liver transplantation, augmentation therapy, gene therapy, peptide therapy and chemical chaperone therapy. In addition to common deficiency variants, there are several rare deficiency variants of α1-AT like Siiyama , Mmalton , Mprocida, Mheerlen, Mmineral springs, Mnichinan, Pduarte, Wbethesda Zaugsberg, and Zbristol. In Siiyama , Mmalton , Mnichinan and Zaugsberg, the β-sheet A is present in an open state, therefore these variants readily undergo polymerization and consequently show aggregation in the hepatocytes. In Mprocida, Mheerlen, Mmineral springs, Pduarte and Wbethesda the conformation is altered significantly, therefore these variants become conformationally less stable and thereby undergo intracellular proteolysis. These rare genetic variants show lungs and / or liver disease. There are several null variants of α1-AT that are not detected either at the stage of transcription or translation. The examples of some of the null variants are QOcardiff, QOhong kong, QOgranite falls, QObellingham, QOmattawa, QObolton, and QOludwigshafen. The molecular basis of deficiency of these variants also form the theme of this review.

Coupling of structural flexibility and biological function is an essential feature of proteins. The role of relative domain movements in enzyme function has been evidenced in many cases. However, the way of communication between protein domains and its manifestation in their movements as well as in the biological function are rarely delineated. In this review we summarize comprehensive studies with a typical hinge-bending two-domain enzyme, 3-phosphoglycerate kinase. A possible mechanism is proposed by which the two substrates that bind to different domains trigger the operation of the molecular hinges, located in the interdomain region. Various crystal structures of the enzyme have been determined with different relative domain positions, suggesting that domain closure brings the two substrates together for the catalysis. Substrate-caused conformational changes in the binary and the ternary complexes have been tested with the solubilized enzyme using physical methods, such as differential scanning calorimetry, small angle X-ray scattering and infrared spectroscopy. The results indicated the existence of strong cooperativity between the two domains and that the presence of both substrates is necessary for the domain closure. Comparison of the atomic contacts in the structures has led to selection of conserved side-chains, which may be involved in the domain movement. On this basis a hypothesis was put forward about the molecular mechanism of interdomain co-operation. Enzyme kinetic, ligand binding and small angle X-ray scattering studies with various site-directed mutants have confirmed this hypothesis. Namely, a special H-bonding network (a double molecular switch) seems to be responsible for operation of the main molecular hinge at the β-strand L under the concerted action of both substrates.

Orexin A (OXA) and orexin B (OXB) are recently discovered neuropeptides that appear to play a role in various distinct functions such as arousal and the sleep-wake cycle as well as on appetite and regulation of feeding and energy homeostasis. Orexins were first described as neuropeptides expressed by a specific population of neurons in the lateral hypothalamic area, a region classically implicated in feeding behaviour. Orexin neurons project to numerous brain regions, where orexin receptors have been shown to be widely distributed: both OXA and OXB act through two subtypes of receptors (OX1R and OX2R) that belong to the G protein-coupled superfamily of receptors. Growing evidence indicates that orexins act in the central nervous system also to regulate gastrointestinal functions: animal studies have indeed demonstrated that centrally-injected orexins or endogenously released orexins in the brain stimulates gastric secretion and influence gastrointestinal motility. The subsequent identification of orexins and their receptors in the enteric nervous system (including the myenteric and the submucosal plexuses) as well as in mucosa and smooth muscles has suggested that these neuropeptides may also play a local action. In this view, emerging studies indicate that orexins also exert region-specific contractile or relaxant effects on isolated gut preparations. The aim of the proposed review is to summarize both centrallyand peripherally-mediated actions of orexins on gastrointestinal functions and to discuss the related physiological role on the basis of the most recent findings.

The last five years have seen a dramatic increase in the number of membrane protein structures. The vast majority of these 191 unique structures are of membrane proteins from prokaryotic sources. Whilst these have provided unprecedented insight into the mechanism of action of these important molecules our understanding of many clinically important eukaryotic membrane proteins remains limited by a lack of high resolution structural data. It is clear that novel approaches are required to facilitate the structural characterization of eukaryotic membrane proteins. Here we review some of the techniques developed recently which are having a major impact on the way in which structural studies of eukaryotic membrane proteins are being approached. Several different high throughput approaches have been designed to identify membrane proteins most suitable for structural studies. One approach is to screen large numbers of related or non-related membrane proteins using GFP fusion proteins. An alternative involves generating large numbers of mutants of a single protein with a view to obtaining a fully functional but highly stable membrane protein. These, and other novel techniques that aim to facilitate the production of membrane protein likely to yield well-diffracting crystals are described.

Prion protein (PrP) gene encodes cellular PrP (PrPC), a glycosylphosphatidylinositol (GPI)-anchored cell membrane protein indispensable for infections of prion, which causes Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Although PrPC is known to be converted into an abnormal isoform (PrPSc) upon prion infection and play an important role in prion diseases, the mechanisms involved remain unclear, partly due to the insolubility of PrPSc, which prevents experimental biochemical and biophysical analyses. Recently, with improvements in computer power and methods, computer analyses have been contributing more to prion studies. A comparison of PrP gene sequences revealed mutations and polymorphisms in the open reading frame (ORF) of the human PrP gene related to prion diseases. In contrast, little mutations or polymorphisms related to susceptibility to BSE were found in the ORF of the bovine PrP gene, though relationships between insertion/deletion (Ins/Del) polymorphisms of the PrP gene promoter and susceptibility to BSE have been found. Our results have shown that the specific protein 1 (Sp1) plays important role in the activity of PrP gene promoter, which is influenced by polymorphisms in the Sp1 binding sites. The potential structural dynamics of PrP have been simulated by computational methods such as molecular dynamics (MD) and quantum mechanics (QM). The proposed mechanisms of conversion have revealed new insights in prion diseases. In this review, we will introduce the gene structure, polymorphisms, and potential structural dynamics of PrP revealed by basic and advanced computational analyses. The possible contribution of these methods to elucidation of the pathogenicity of prion diseases and functions of PrPC is discussed.