Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

Human ζ-crystallin is a Zn²⁺-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,β-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn²⁺-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.     

What’s new in the renin-angiotensin system?

Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of -helices, is characterised by a core channel that has a constriction approximately 10 Å from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.     

The interface of protein-protein complexes: Analysis of contacts and prediction of interactions

Specific protein-protein interactions are essential for cellular functions. Experimentally determined three-dimensional structures of protein-protein complexes offer the possibility to characterize binding interfaces in terms of size, shape and packing density. Comparison with crystal-packing interfaces representing nonspecific protein-protein contacts gives insight into how specific binding differs from nonspecific low-affinity binding. An overview is given on empirical structural rules for specific protein-protein recognition derived from known complex structures. Although single parameters such as interface size, shape or surface complementary show clear trends for different interface types, each parameter alone is insufficient to fully distinguish between specific versus crystal-packing contacts. A combination of interface parameters is, however, well suited to characterize a specific interface. This knowledge provides us with the essential ingredients that make up a specific protein recognition site. It is also of great value for the prediction of protein binding sites and for the evaluation of predicted complex structures. Received 1 October 2007; received after revision 9 November 2007; accepted 9 November 2007     

The role of key residues in structure, function, and stability of cytochrome-c

Cytochrome-c (cyt-c), a multi-functional protein, plays a significant role in the electron transport chain, and thus is indispensable in the energy-production process. Besides being an important component in apoptosis, it detoxifies reactive oxygen species. Two hundred and eighty-five complete amino acid sequences of cyt-c from different species are known. Sequence analysis suggests that the number of amino acid residues in most mitochondrial cyts-c is in the range 104?±?10, and amino acid residues at only few positions are highly conserved throughout evolution. These highly conserved residues are Cys14, Cys17, His18, Gly29, Pro30, Gly41, Asn52, Trp59, Tyr67, Leu68, Pro71, Pro76, Thr78, Met80, and Phe82. These are also known as “key residues”, which contribute significantly to the structure, function, folding, and stability of cyt-c. The three-dimensional structure of cyt-c from ten eukaryotic species have been determined using X-ray diffraction studies. Structure analysis suggests that the tertiary structure of cyt-c is almost preserved along the evolutionary scale. Furthermore, residues of N/C-terminal helices Gly6, Phe10, Leu94, and Tyr97 interact with each other in a specific manner, forming an evolutionary conserved interface. To understand the role of evolutionary conserved residues on structure, stability, and function, numerous studies have been performed in which these residues were substituted with different amino acids. In these studies, structure deals with the effect of mutation on secondary and tertiary structure measured by spectroscopic techniques; stability deals with the effect of mutation on T _m (midpoint of heat denaturation), ?G _D (Gibbs free energy change on denaturation) and folding; and function deals with the effect of mutation on electron transport, apoptosis, cell growth, and protein expression. In this review, we have compiled all these studies at one place. This compilation will be useful to biochemists and biophysicists interested in understanding the importance of conservation of certain residues throughout the evolution in preserving the structure, function, and stability in proteins.     

The role of bystin in embryo implantation and in ribosomal biogenesis

Human bystin was identified as a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. Although the trophinin gene is unique to mammals, the bystin gene (BYSL) is conserved across eukaryotes. Recent studies show that bystin plays a key role during the transition from silent trophectoderm to an active trophoblast upon trophinin-mediated cell adhesion. Bystin gene knockout and knockdown experiments demonstrate that bystin is essential for embryonic stem cell survival and trophectoderm development in the mouse. Furthermore, biochemical analysis of bystin in human cancer cells and mouse embryos indicates a function in ribosomal biogenesis, specifically in processing of 18S RNA in the 40S subunit. Strong evidence that BYSL is a target of c-MYC is consistent with a role for bystin in rapid protein synthesis, which is required for actively growing cells. Received 30 June 2007; received after revision 7 August 2007; accepted 29 August 2007     

Mitochondrial association of alpha-synuclein causes oxidative stress

α-Synuclein is a neuron-specific protein that contributes to the pathology of Parkinson’s disease via mitochondria-related mechanisms. The present study investigated possible interaction of α-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing α-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that α-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of α-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by α-synuclein. Received 27 December 2007; received after revision 7 February 2008; accepted 8 February 2008     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Monogenic determinants of familial Alzheimer's disease: presenilin-2 mutations

Presenilin-2 (PS2) is one of three genes [amyloid precursor protein (APP), presenilin-1 (PS1) and PS2] shown to cause familial Alzheimer's disease (FAD), and is highly homologous to PS1. Currently demonstrated functions of PS2 include interactions with APP and Aβ, and participation in apoptotic pathways. PS2 FAD mutations influence APP processing in a manner predicted to promote amyloid formation and also enhance the proapoptotic effect of wild-type PS2. Other possible functions of PS2 are related to its homology to Notch pathway genes in Caenorhabditis elegans, suggesting it may have a developmental role. PS2-associated AD is the most reminiscent of the sporadic form of the disease in terms of older age of onset and longer disease duration. Since PS2 mutations are incompletely penetrant and age of onset in carriers is highly variable (40 – 88 years), elucidation of PS2 mechanisms may reveal factors which modify AD and are therapeutically relevant to sporadic AD.     

(In)discrete charm of the polyembryony: Evolution of embryo cloning

Polyembryonic development, where multiple embryos are formed from a single zygote, evolved at least 15 times in six different phyla in animals. The mechanisms leading to polyembryony and the forces that shaped the evolution of the polyembryonic developmental program have remained poorly understood. Recent studies of the polyembryonic development in the endoparasitic wasp Copidosoma floridanum have revealed that the evolution of polyembryony is associated with the evolution of developmental novelties such as total cleavage, early specification of embryonic and extra-embryonic fates, and a specific cell proliferation phase. These changes cumulatively result in the formation of thousands of embryos from a single egg. Laser ablation studies and analysis of early cell fate specification have revealed that a single blastomere representing the progenitor of the primordial germ cell regulates the proliferation of the embryos. We propose that evolutionary changes in cell cleavage, cell interactions, and the cell-differentiation program, reminiscent of interactions between the germinal stem cell and stem cell niche in fly ovaries, underlies the evolution of polyembryony. Received 30 January 2007; received after revision 21 June 2007; accepted 11 July 2007     

Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same α β dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992), Science 255: 54–63]. The implications of this model and the relevant experiments will be reviewed here. Received 27 April 1998; received after revision 17 July 1998; accepted 10 August 1998     

Hydroxyproline-rich glycoproteins in plant reproductive tissues: structure, functions and regulation

The plant reproductive process of pollination involves a series of interactions between the male gametophyte (the pollen grain or pollen tube) and extracellular matrix (ECM) molecules secreted by different cell types along the pollen tube growth pathway in the female organ, the pistil. These interactions are believed to signal and regulate the pollen tube growth process to effect successful delivery of the sperm cells to the ovules where fertilization takes place. Hydroxyproline-rich glycoproteins secreted by plant cells are believed to play a broad range of functions, ranging from providing structural integrity to mediating cell-cell interactions and communication. The pistil and pollen tube ECM is enriched in these highly glycosylated proteins. Our discussions here will focus on a number of these proteins for which most information has been available, from Nicotiana tabacum, its self-incompatible relative N. alata, and Zea mays. In addition, the regulation of the synthesis and glyco-modification of one of these proteins, TTS (transmitting tissue-specific) protein from N. tabacum will be discussed in the light of how differential glycosylation may be used to regulate molecular interactions within the ECM.     

Cyanovirin-N: a sugar-binding antiviral protein with a new twist

Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N. RID="*" ID="*"Corresponding author.     

Interactions of the cell adhesion molecule nectin with transmembrane and peripheral membrane proteins for pleiotropic functions

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin α_vβ₃ and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions. Received 27 June 2007; received after revision 14 August 2007; accepted 12 September 2007     

Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism

The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K_m values ranged from 0.05 to 0.3 μM and k_cat values from 2.3 to 17.6 min⁻¹, while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K_m values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations. Received 12 October 2006; received after revision 6 December 2006; accepted 8 January 2007     

Role of heregulin in human cancer

Heregulin (HRG) is a soluble secreted growth factor, which, upon binding and activation of ErbB3 and ErbB4 transmembrane receptor tyrosine kinases, is involved in cell proliferation, invasion, survival and differentiation of normal and malignant tissues. The HRG gene family consists of four members: HRG-1, HRG-2, HRG-3 and HRG-4, of which a multitude of different isoforms are synthesized by alternative exon splicing, showing various tissue distribution and biological activities. Disruption of the physiological balance between HRG ligands and their ErbB receptors is implicated in the formation of a variety of human cancers. The general mechanisms involved in HRG-induced tumorigenesis is discussed. Received 8 March 2007; received after revision 6 May 2007; accepted 9 May 2007     

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Unveiling molecular mechanisms of bacterial surface proteins: <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> as a model organism for structural studies

Bacteria present a variety of molecules either on their surface or in a cell-free form. These molecules take part in numerous processes in the interactions with their host, with its tissues and other molecules. These molecules are essential to bacterial pathogenesis either during colonization or the spread/invasion stages, and most are virulence factors. This review is focused on such molecules using Streptococcus pneumoniae, a Gram-positive bacterium, as an example. Selected surface proteins are introduced, their structure described, and, whenever available, their mechanisms of function on an atomic level are explained. Such mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed. Elucidation of molecular mechanisms of virulence factors is essential for the understanding of bacteria and their functional properties. Structural biology appears pivotal for these studies, as structural and mechanistic insights facilitate rational approach to the development of new treatments. Received 12 March 2007; received after revision 28 June 2007; accepted 18 July 2007     

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     

Die Veränderungen der Succinodehydrase-Aktivität in Detroit-6 (VA) Zellen,infolge der Infektion mit demHepatitis-infectiosa-Virus

Summary The inhibitory action ofHepatitis infectiosa virus on the SDH activity of Detroit-6 (VA) cell lines was investigated. The full inhibition of the SDH activity took place at the end of the third day after the infection. As this phenomenon precedes the cytopathogenetic effect of viral infection, it may be of some help in the early detection of the infection.     

Interaction of aging-associated signaling cascades: Inhibition of NF-κB signaling by longevity factors FoxOs and SIRT1

Research on aging in model organisms has revealed different molecular mechanisms involved in the regulation of the lifespan. Studies on Saccharomyces cerevisiae have highlighted the role of the Sir2 family of genes, human Sirtuin homologs, as the longevity factors. In Caenorhabditis elegans, the daf-16 gene, a mammalian homolog of FoxO genes, was shown to function as a longevity gene. A wide array of studies has provided evidence for a role of the activation of innate immunity during aging process in mammals. This process has been called inflamm-aging. The master regulator of innate immunity is the NF-κB system. In this review, we focus on the several interactions of aging-associated signaling cascades regulated either by Sirtuins and FoxOs or NF-κB signaling pathways. We provide evidence that signaling via the longevity factors of FoxOs and SIRT1 can inhibit NF-κB signaling and simultaneously protect against inflamm-aging process. Received 4 October 2007; received after revision 7 November 2007; accepted 9 November 2007