The eosinophil ribonucleases

The eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3) are two closely related proteins with intriguing functional and evolutionary properties. While both EDN and ECP maintain the structural and catalytic residues typical of the RNase A superfamily, the role of ribonuclease activity in the physiologic function of these proteins remains unclear. The biochemistry and physiology of EDN, ECP and the recently discovered ribonuclease k6 (RNase 6) will be reviewed in this chapter.     

Molecular modeling of mammalian cytochrome P450s

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure, models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s. Received 28 September 1999; received after revision 25 November 1999; accepted 31 December 1999     

The angiogenins

The angiogenic and other biological functions of the angiogenins, members of the pancreatic RNase superfamily of proteins, are reviewed in the context of their primary and tertiary structures. The ribonucleolytic activity and interactions with the placental ribonuclease inhibitor have seen much study in the last few years. The mechanism of the angiogenic activity of angiogenin has recently been postulated as involving multiple interactions with other proteins through specific regions on the molecular surface of angiogenin. These molecular partners include heparin, plasminogen, elastase, angiostatin, actin and most importantly a 170-kilodalton receptor on subconfluent endothelial cells. The existence of the latter receptor was established in conjunction with a mitogenic activity of angiogenin on subconfluent cells. The levels of angiogenin in various physiological and disease states are summarized, including various studies on pregnancy and angiogenin. Correlations are seen between states of enhanced angiogenesis and angiogenin levels. An overview of the relationship of angiogenin and the other RNases of the superfamily showed that their genes all are in relative close proximity on human chromosome 14. Examination of the many expressed sequence tags published in the public databanks, for angiogenin and the other RNases, revealed that angiogenin and RNase-4 (the most evolutionarily conserved RNase), share various identical 5′-untranslated regions on their sets of messenger RNAs, suggesting that their genes are in very close proximity on chromosome 14 and that they are products of differential splicing. This in turn suggests that, in both humans and mice, expression of these two proteins is under identical control, with obvious implications for their biological activities. The evolutionary history of the angiogenins is examined briefly on the basis of the protein sequences of the human, rabbit, pig, two bovine and four mouse angiogenins, and two mouse angiogenin pseudogene sequences. The discrepancy between the conventional requirement for conservatism in structure to allow multimolecule interactions, and the actual fast-changing sequence of the angiogenins, in concert with the wide-ranging activity even in birds, of human angiogenin, is discussed.     

Are Rab proteins the link between Golgi organization and membrane trafficking?

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Saccharomyces cerevisiae, the Golgi cisternae are physically separated from one another, while in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells, many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi-associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18, and Rab43. In yeast, these include Ypt1, Ypt32, and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport.     

Gelsolin superfamily proteins: key regulators of cellular functions

Cytoskeletal rearrangement occurs in a variety of cellular processes and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins control actin organization by severing filaments, capping filament ends and nucleating actin assembly [1]. Gelsolin is the founding member of this family, which now contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. In addition to their respective role in actin filament remodeling, these proteins have some specific and apparently non-overlapping particular roles in several cellular processes, including cell motility, control of apoptosis and regulation of phagocytosis (summarized in table 1). Evidence suggests that proteins belonging to the gelsolin superfamily may be involved in other processes, including gene expression regulation. This review will focus on some of the known functions of the gelsolin superfamily proteins, thus providing a basis for reflection on other possible and as yet incompletely understood roles for these proteins.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

The Ras family of GTPases in cancer cell invasion

The ability of tumoral cells to invade surrounding tissues is a prerequisite for metastasis. This is the most life-threatening event of tumor progression, and so research is intensely focused on elucidating the mechanisms responsible for invasion and metastasis. The Ras superfamily of GTPases comprises several subfamilies of small GTP-binding proteins whose functions include the control of proliferation, differentiation, and apoptosis, as well as cytoskeleton organization. The development of metastasis is a multistep process that requires coordinated activation of proliferation, motility, changes in normal cell-to-cell and cell-to-substrate contacts, degradation of extracellular matrix, inhibition of apoptosis, and adaptation to an inappropriate tissue environment. Several members of the Ras superfamily of proteins have been implicated in these processes. The present review summarizes the current knowledge in this field.     

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.     

Ribonuclease 4, an evolutionarily highly conserved member of the superfamily

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a K _d of 4 × 10⁻¹⁵ M.     

Metazoan evolution of the armadillo repeat superfamily

The superfamily of armadillo repeat proteins is a fascinating archetype of modular-binding proteins involved in various fundamental cellular processes, including cell–cell adhesion, cytoskeletal organization, nuclear import, and molecular signaling. Despite their diverse functions, they all share tandem armadillo (ARM) repeats, which stack together to form a conserved three-dimensional structure. This superhelical armadillo structure enables them to interact with distinct partners by wrapping around them. Despite the important functional roles of this superfamily, a comprehensive analysis of the composition, classification, and phylogeny of this protein superfamily has not been reported. Furthermore, relatively little is known about a subset of ARM proteins, and some of the current annotations of armadillo repeats are incomplete or incorrect, often due to high similarity with HEAT repeats. We identified the entire armadillo repeat superfamily repertoire in the human genome, annotated each armadillo repeat, and performed an extensive evolutionary analysis of the armadillo repeat proteins in both metazoan and premetazoan species. Phylogenetic analyses of the superfamily classified them into several discrete branches with members showing significant sequence homology, and often also related functions. Interestingly, the phylogenetic structure of the superfamily revealed that about 30 % of the members predate metazoans and represent an ancient subset, which is gradually evolving to acquire complex and highly diverse functions.     

Peptides in the mammalian cardiovascular system

Summary Ample immunocytochemical evidence is now available demonstrating that several peptides are present in the mammalian cardiovascular system where they are localised to nerve fibres and myocardial cells. The neuropeptides (neuropeptide Y, calcitonin gene-related peptide, tachykinins and vasoctive intestinal polypeptide) are localised to large secretory vesicles in subpopulations of afferent or efferent nerves supplying the heart and vasculature of several mammals, including man. Although they often exert potent pharmacological effects on the tissues in which they occur their physiological significance has still to be established. They may act directly via specific receptors and/or indirectly by influencing the release and action of other cardiovascular transmitters. In marked contrast, atrial natriuretic peptide is produced by cardiac myocytes and considered to act as a circulating hormone.     

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present. Received 26 October 2007; accepted 23 November 2007     

Peptides in the mammalian cardiovascular system

Ample immunocytochemical evidence is now available demonstrating that several peptides are present in the mammalian cardiovascular system where they are localised to nerve fibres and myocardial cells. The neuropeptides (neuropeptide Y, calcitonin gene-related peptide, tachykinins and vasoactive intestinal polypeptide) are localised to large secretory vesicles in subpopulations of afferent or efferent nerves supplying the heart and vasculature of several mammals, including man. Although they often exert potent pharmacological effects on the tissues in which they occur their physiological significance has still to be established. They may act directly via specific receptors and/or indirectly by influencing the release and action of other cardiovascular transmitters. In marked contrast, atrial natriuretic peptide is produced by cardiac myocytes and considered to act as a circulating hormone.     

Involvement of ribonuclease in the interactions of macrophages and fibroblasts in experimental silicosis

Summary Decreased ribonuclease activity in the supernatant from silica-treated macrophages is associated with the enhanced protein synthesis in granulation-tissue slices incubated in this supernatant, and with the decreased degradation of polysomes in granuloma slices and of polysomes isolated from the granulation tissue. The phagocytized silica particles adsorb ribonuclease and perhaps other proteins and thus remove them from the macrophage supernatant.For financial support we are grateful to the Association of Finnish Life Assurance Companies and to the Medical Research Council in Finland.