Recent progress in understanding the diversity of the human ov-serpin/clade B serpin family

The inhibitory mechanism against proteases is important in the maintenance of homeostasis or health in the body. The human ovalbumin serpin (ovserpin)/ clade B serpin family is one group of the human serpins, a family of serine protease inhibitors. They have acquired diversity in the profiles of target proteases, inhibitory mechanisms, and localization patterns during their evolution. Most serpins target serine proteases, however, some ov-serpins target only cysteine proteases or both serine and cysteine proteases and furthermore, several ov-serpins do not possess inhibitory activities. Although the ov-serpins act primarily as intracellular serpins, some show extracellular and nuclear localizations. Such diversity enables the ov-serpins to play multiple physiological roles in the body. Recent analyses have revealed that the functions of human ov-serpins are more diversified than we previously knew. In this article, we describe recent progress in our understanding of how the human ov-serpin/clade B serpin family demonstrates diversity.     

The role of serpins in the surveillance of the secretory pathway

Serpins (serine protease inhibitors) constitute a class of proteins with an unusually wide spectrum of different functions at extracellular sites and within the nucleocytoplasmic compartment that extends from protease inhibition to hormone transport and regulation of chromatin organization. Recent investigations reveal a growing number of serpins acting in secretory pathway organelles, indicating that they are not simply cargo destined for export, but fulfill distinct roles within the classical organelle-coupled trafficking system. These findings imply that some serpins are part of a quality control system that monitors the export and possibly import routes of eukaryotic cells. The molecular targets of these serpins are often unknown, opening new avenues for future research.     

The DnaK/ClpB chaperone system from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Proteins of thermophilic organisms are adapted to remain well structured and functional at elevated temperatures. Nevertheless like their 'cousins' that reside at medium temperatures, they require the assistance of molecular chaperones to fold properly and prevent aggregation. This review compares structural and functional properties of the DnaK/ClpB systems of Thermus thermophilus and, mainly, Escherichia coli (Dnak_Tth and Dnak_Eco). Many elemental properties of these systems remain conserved. However, in addition to a general increase of the thermal stability of its components, the Dnak_Tth system shows profound differences in its regulation, and genetic as well as oligomeric organization. Whether these differences are unique or represent general strategies of adaptation to life at elevated temperatures remains to be clarified. RID="*" ID="*"Corresponding author.     

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, <Emphasis Type="Italic">Calyptogena</Emphasis>

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33–42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei. Received 13 May 2003; received after revision 5 June 2003; accepted 12 June 2003     

The vault complex

Vaults are large ribonucleoprotein particles found in eukaryotic cells. They are composed of multiple copies of a M _r 100,000 major vault protein and two minor vault proteins of M _r 193,000 and 240,000, as well as small untranslated RNAs of 86–141 bases. The vault components are arranged into a highly characteristic hollow barrel-like structure of 35 × 65 nm in size. Vaults are predominantly localized in the cytoplasm where they may associate with cytoskeletal elements. A small fraction of vaults are found to be associated with the nucleus. As of yet, the precise cellular function of the vault complex is unknown. However, their distinct morphology and intracellular distribution suggest a role in intracellular transport processes. Here we review the current knowledge on the vault complex, its structure, components and possible functions.Received 23 January 2003; received after revision 13 March 2003; accepted 26 March 2003     

Molecular neurophysiology of taste in <Emphasis Type="Italic">Drosophila</Emphasis>

The recent identification of candidate receptor genes for sweet, umami and bitter taste in mammals has opened a door to elucidate the molecular and neuronal mechanisms of taste. Drosophila provides a suitable system to study the molecular, physiological and behavioral aspects of taste, as sophisticated molecular genetic techniques can be applied. A gene family for putative gustatory receptors has been found in the Drosophila genome. We discuss here current knowledge of the gustatory physiology of Drosophila. Taste cells in insects are primary sensory neurons whereupon each receptor neuron responds to either sugar, salt or water. We found that particular tarsal gustatory sensilla respond to bitter compounds. Electrophysiological studies indicate that gustatory sensilla on the labellum and tarsi are heterogeneous in terms of their taste sensitivity. Determination of the molecular bases for this heterogeneity could lead to an understanding of how the sensory information is processed in the brain and how this in turn is linked to behavior.Received 12 May 2003; received after revision 9 June 2003; accepted 13 June 2003     

Vaspin inhibits kallikrein 7 by serpin mechanism

The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin–hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.     

The <Emphasis Type="Italic">Adhesion</Emphasis> GPCRs: A unique family of G protein-coupled receptors with important roles in both central and peripheral tissues

G protein-coupled receptors (GPCRs) are a diverse superfamily of membrane-bound receptors. The second largest subgroup of GPCRs, the Adhesion GPCRs, has 33 members in humans. Phylogenetic analysis of the entire repertoire of the seven transmembrane- domain (7TM) regions of GPCRs shows that the Adhesion GPCRs form a distinct family. Adhesion GPCRs are characterised by (1) long N termini with multiple functional domains often found in other proteins such as tyrosine kinases, integrins and cadherins, (2) highly complex genomic structure with multiple introns and splice variants and (3) a 7TM region that has no clear similarities with 7TM from other GPCRs. Several Adhesion GPCRs are known to have a role in the immune system but it is becoming more evident that many have important roles in the CNS. We speculate that the overall structural construction of the Adhesion GPCRs allows them to participate in different types of cell guidance. Received 8 February 2007; received after revision 21 March 2007; accepted 25 April 2007     

Structural insights into the multiple functions of protein C inhibitor

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport. Received 1 July 2008; received after revision 16 August 2008; accepted 22 August 2008     

The role of proteolytic processing in the morphogenesis of virus particles

Proteinases are encoded by many RNA viruses, all retroviruses and several DNA viruses. They play essential roles at various stages in viral replication, including the coordinated assembly and maturation of virions. Most of these enzymes belong to one of three (Ser, Cys or Asp) of the four major classes of proteinases, and have highly substrate-selective and cleavage specific activities. They can be thought of as playing one of two general roles in viral morphogenesis. Structural proteins are encoded by retroviruses and many RNA viruses as part of large polyproteins. Their proteolytic release is a prerequisite to particle assembly; consequent structural rearrangement of the capsid domains serves to regulate and direct association and assembly of capsid subunits. The second general role of proteolysis is in assembly-dependent maturation of virus particles, which is accompanied by the acquisition of infectivity.     

Peroxiredoxin 2 (<Emphasis Type="Italic">PRDX2</Emphasis>), an antioxidant enzyme,is underexpressed in Down syndrome fetal brains

Suppression subtractive hybridization performed on Down syndrome (DS) versus control fetal brains revealed differential expression of peroxiredoxin 2 (PRDX2), mapped at 13q12. Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. The under-expression of PRDX2 observed in DS samples was confirmed by realtime PCR (0.73-fold). To test whether decreased expression is associated with enhanced sensitivity of DS neurons to reactive oxygen species, we down-regulated PRDX2 through stable transfections of SH-SY5Y neuroblastoma cells with antisense contructs of the complete PRDX2 coding sequence. In addition, we over-expressed SOD1 and compared the effects of the two genes on cell viability. Cells transfected with either construct showed similar sensitivity to oxidative stress in addition to increased apoptosis under basal conditions and after treatment with oxidative cytotoxic agents. This suggests that the decreased expression of PRDX2 may contribute to the altered redox state in DS at levels comparable to that of the increased expression of SOD1.Received 4 February 2003; received after revision 31 March 2003; accepted 25 April 2003     

Genetics of early mammalian folliculogenesis

Early ovarian folliculogenesis begins with the breakdown of germ cell clusters and formation of primordial follicles. Primordial follicles are the smallest ovarian follicle units continuously recruited to grow into primary and more advanced ovarian follicles. Genes expressed in the germ cells such as Figla, Nobox, Kit and Ntrk2, as well as genes expressed in the surrounding somatic cells such as Foxl2, Kitl and Ngf, play critical functions during early folliculogenesis. Transgenic mice continue to provide important insights into the genetic pathways that regulate early mammalian folliculogenesis. Genes critical in early folliculogenesis are important determinants of reproductive life span and represent candidate genes for human ovarian failure. Received 25 August 2005; received after revision 18 October 2005; accepted 21 November 2005     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudopepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S₄ through S₃′ subsites. Hydrophobic and aromatic amino acids are preferred at the P₁ and P₁′ positions. Interactions at additional subsites are important in some cases, for example with cleavage of κ-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones. Received 25 May 2001; received after revision 27 August 2001; accepted 30 August 2001     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Neuroprotective effects of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> extract

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this review, we summarize the experimental results on the mechanism of neuroprotection induced by standardized extract of Ginkgo biloba leaves (EGb 761) and its constituents. The effects described mostly in animals include those on cerebral blood flow, neurotransmitter systems, cellular redox state and nitric oxide level. Furthermore, we discuss the current status of clinical trials as well as undesired side effects of EGb 761.Received 21 November 2002; received after revision 8 March 2003; accepted 17 March 2003     

A human homolog of the yeast gene encoding tRNA 2′-phosphotransferase: cloning,characterization and complementation analysis

The Saccharomyces cerevisiae TPT1 gene plays a role in removing the 2-phosphate from ligated tRNA during the maturation of pre-tRNA. Here we reported the cloning and characterization of the human TRPT1 gene as a homolog of yeast TPT1. The TRPT1 gene is located at human chromosome 11q13 and encodes a polypeptide of 253 amino acids. BLAST searches with its amino acid sequence revealed the ubiquitous occurrence of TRPT1 homologs and their functional relationships with the presence of the DUF60/KptA domain. Northern analysis demonstrated that the gene is primarily expressed in heart and skeletal muscle, with lower or undetectable levels in other tissues studied. A plasmid-shuffling experiment showed that the human TRPT1 gene could complement the tpt1 mutation in S. cerevisiaeReceived 19 March 2003; received after revision 25 April 2003; accepted 22 May 2003     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> innate immunity: an emerging role for peptidoglycan recognition proteins in bacteria detection

Over the past years, parallel studies conducted in mammals and flies have emphasized the existence of common mechanisms regulating the vertebrate and invertebrate innate immune systems. This culminated in the discovery of the central role of the Toll pathway in Drosophila immunity and in the implication of Toll-like receptors (TLRs)/interleukin-1(IL-1) in the mammalian innate immune response. In spite of clear similarities, such as shared intracellular pathway components, important divergences are expected between the two groups, whose last common ancestor lived more than half a billion years ago. The most obvious discrepancies lie in the mode of activation of the signalling receptors by microorganisms. In mammals, TLRs are part of protein complexes which directly recognize microbe-associated patterns, whereas Drosophila Toll functions like a classical cytokine receptor rather than a pattern recognition receptor. Recent studies demonstrate that members of the evolutionarily conserved peptidoglycan recognition protein family play an essential role in microbial sensing during immune response of Drosophila.Received 26 June 2003; received after revision 29 July 2003; accepted 25 August 2003     

Expression of multidrug resistance (mdr) gene(s) in primary lymphoid organs of chicken immune system during embryonic development

The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described. The present report evaluates the expression of themdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching. Results show that the thymic cells are positive from day 12 to the end of the observation period. In contrast,mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life. Possible relationships between the expression ofmdr and the development of T and B lymphocytes are discussed.