Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.     

Assembling an ion channel: ORF 3a from SARS‐CoV

Protein 3a is a 274 amino acid polytopic channel protein with three putative transmembrane domains (TMDs) encoded by severe acute respiratory syndrome corona virus (SARS‐CoV). Synthetic peptides corresponding to each of its three individual transmembrane domains (TMDs) are reconstituted into artificial lipid bilayers. Only TMD2 and TMD3 induce channel activity. Reconstitution of the peptides as TMD1 + TMD3 as well as TMD2 + TMD3 in a 1 : 1 mixture induces membrane activity for both mixtures. In a 1 : 1 : 1 mixture, channel like behavior is almost restored. Expression of full length 3a and reconstitution into artificial lipid bilayers reveal a weak cation selective (P_K ≈ 2 P_Cl) rectifying channel. In the presence of nonphysiological concentration of Ca‐ions the channel develops channel activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99:628–635, 2013.     

Structural elucidation of transmembrane transporter protein bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy

Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds.     

Modes of acrosin functioning during fertilization

Mammalian fertilization is a complex process that involves gamete recognition, penetration, and fusion. Biochemical studies that identified the role of acrosome components during sperm–ova interaction especially the zona pellucida (ZP) provided major advances in sperm cell biology. Acrosin (a typical serine protease) functions during fertilization in several significant ways which include: a) activation of acrosome components, b) secondary binding with the ZP, and c) hydrolysis of the ZP. However, studies using knockout (KO) acrosin-deficient mice cast doubt on the traditional role of acrosin in fertilization. The KO acrosin-deficient mice exhibit normal fecundity except for delayed fertilization. Despite the doubt cast on the traditional role of acrosin by the KO acrosin-deficient mouse studies, acrosin still remains a major protease involved in multiple processes of fertilization. In this review, we assess the functional profile of acrosin and briefly summarize recent findings on proteases involved in fertilization. We propose a refined scheme for the functional role of acrosin in fertilization. We particularly emphasize the role of acrosin in acrosome exocytosis and activation of other acrosome components based on advanced technology like structural X-ray analysis.     

Unraveling microalgal molecular interactions using evolutionary and structural bioinformatics

Microalgae are unicellular microorganisms indispensible for environmental stability and life on earth, because they produce approximately half of the atmospheric oxygen, with simultaneously feeding on the harmful greenhouse gas carbon dioxide. Using gene fusion analysis, a series of five fusion/fission events was identified, that provided the basis for critical insights to their evolutionary history. Moreover, the three-dimensional structures of both the fused and the component proteins were predicted, allowing us to envisage putative protein–protein interactions that are invaluable for the efficient usage, handling and exploitation of microalgae. Collectively, our proposed approach on the five fusion/fission alga protein events contributes towards the expansion of the microalgae knowledgebase, bridging protein evolution of the ancient microalgal species and the rapidly evolving, modern, bioinformatics field.     

Adaptive evolution of tight junction protein claudin-14 in echolocating whales

Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats.     

Comparative genomic analysis and evolution of family-B G protein-coupled receptors from six model insect species

Family-B G protein-coupled receptors (GPCR-Bs) play vital roles in many biological processes, including growth, development and reproduction. However, the evolution and function of GPCR-Bs have been poorly understood in insects.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.