Showing papers in "FEBS Journal in 2004"

Endovanilloids. Putative endogenous ligands of transient receptor potential vanilloid 1 channels.

Abstract: Endovanilloids are defined as endogenous ligands of the transient receptor potential vanilloid type 1 (TRPV1) protein, a nonselective cation channel that belongs to the large family of TRP ion channels, and is activated by the pungent ingredient of hot chilli peppers, capsaicin. TRPV1 is expressed in some nociceptor efferent neurons, where it acts as a molecular sensor of noxious heat and low pH. However, the presence of these channels in various regions of the central nervous system, where they are not likely to be targeted by these noxious stimuli, suggests the existence of endovanilloids. Three different classes of endogenous lipids have been found recently that can activate TRPV1, i.e. unsaturated N-acyldopamines, lipoxygenase products of arachidonic acid and the endocannabinoid anandamide with some of its congeners. To classify a molecule as an endovanilloid, the compound should be formed or released in an activity-dependent manner in sufficient amounts to evoke a TRPV1-mediated response by direct activation of the channel. To control TRPV1 signaling, endovanilloids should be inactivated within a short time-span. In this review, we will discuss, for each of the proposed endogenous ligands of TRPV1, their ability to act as endovanilloids in light of the criteria mentioned above.

Mitochondrial beta-oxidation.

Abstract: Mitochondrial beta-oxidation is a complex pathway involving, in the case of saturated straight chain fatty acids of even carbon number, at least 16 proteins which are organized into two functional subdomains; one associated with the inner face of the inner mitochondrial membrane and the other in the matrix. Overall, the pathway is subject to intramitochondrial control at multiple sites. However, at least in the liver, carnitine palmitoyl transferase I exerts approximately 80% of control over pathway flux under normal conditions. Clearly, when one or more enzyme activities are attenuated because of a mutation, the major site of flux control will change.

New human and mouse microRNA genes found by homology search

Abstract: Conservation of microRNAs (miRNAs) among species suggests that they bear conserved biological functions. However, sequencing of new miRNAs has not always been accompanied by a search for orthologues in other species. I report herein the results of a systematic search for interspecies orthologues of miRNA precursors, leading to the identification of 35 human and 45 mouse new putative miRNA genes. MicroRNA tracks were written to visualize miRNAs in human and mouse genomes on the UCSC Genome Browser. Based on their localization, miRNA precursors can be excised either from introns or exons of mRNAs. When intronic miRNAs are antisense to the apparent host gene, they appear to originate from ill-characterized antisense transcription units. Exonic miRNAs are, in general, nonprotein-coding, poorly conserved genes in sense orientation. In three cases, the excision of an miRNA from a protein-coding mRNA might lead to the degradation of the rest of the transcript. Moreover, three new examples of miRNAs fully complementary to an mRNA are reported. Among these, miR135a might control the stability and/or translation of an alternative form of the glycerate kinase mRNA by RNA interference. I also discuss the presence of human miRNAs in introns of paralogous genes and in miRNA clusters.

Biochemical pharmacology of the vanilloid receptor TRPV1

Abstract: There is mounting evidence that the vanilloid (capsaicin) receptor; transient receptor potential channel, vanilloid subfamily member 1 (TRPV1), is subjected to multiple interacting levels of control. The first level is by reversible phosphorylation catalyzed by intrinsic kinases (e.g. protein kinase A and C) and phosphatases (e.g. calcineurin), which plays a pivotal role in receptor sensitization vs. tachyphylaxis. In addition, this mechanism links TRPV1 to intracellular signaling by various important endogenous as well as exogenous substances such as bradykinin, ethanol, nicotin and insulin. It is not clear, however, whether phosphorylation per se is sufficient to liberate TRPV1 under the inhibitory control of phosphatydylinositol-4,5-bisphosphate. The second level of control is by forming TRPV1 heteromers and their association with putative regulatory proteins. The next level of regulation is by subcellular compartmentalization. The membrane form of TRPV1 functions as a nonselective cation channel. On the endoplasmic reticulum, TRPV1 is present in two differentially regulated forms, one of which is inositol triphosphate-dependent whereas the other is not. These three TRPV1 compartments provide a versatile regulation of intracellular Ca2+ levels. Last, there is a complex and poorly understood regulation of TRPV1 activity via control of gene expression. Factors that downregulate TRPV1 expression include vanilloid treatment and growth factor (notably, nerve growth factor) deprivation. By contrast, TRPV1 appears to be upregulated during inflammatory conditions. Interestingly, following experimental nerve injury and in animal models of diabetic neuropathy TRPV1 is present on neurons that do not normally express TRPV1. Combined, these findings imply an important role for aberrant TRPV1 expression in the development of neuropathic pain and hyperalgesia. In humans, disease-related changes in TRPV1 expression have already been described (e.g. inflammatory bowel disease and irritable bowel syndrome). The mechanisms that regulate TRPV1 gene expression under pathological conditions are unknown but a better understanding of these pathways has obvious implications for rational drug development.

The principle of flux minimization and its application to estimate stationary fluxes in metabolic networks.

Abstract: Cellular functions are ultimately linked to metabolic fluxes brought about by thousands of chemical reactions and transport processes. The synthesis of the underlying enzymes and membrane transporters causes the cell a certain 'effort' of energy and external resources. Considering that those cells should have had a selection advantage during natural evolution that enabled them to fulfil vital functions (such as growth, defence against toxic compounds, repair of DNA alterations, etc.) with minimal effort, one may postulate the principle of flux minimization, as follows: given the available external substrates and given a set of functionally important 'target' fluxes required to accomplish a specific pattern of cellular functions, the stationary metabolic fluxes have to become a minimum. To convert this principle into a mathematical method enabling the prediction of stationary metabolic fluxes, the total flux in the network is measured by a weighted linear combination of all individual fluxes whereby the thermodynamic equilibrium constants are used as weighting factors, i.e. the more the thermodynamic equilibrium lies on the right-hand side of the reaction, the larger the weighting factor for the backward reaction. A linear programming technique is applied to minimize the total flux at fixed values of the target fluxes and under the constraint of flux balance (= steady-state conditions) with respect to all metabolites. The theoretical concept is applied to two metabolic schemes: the energy and redox metabolism of erythrocytes, and the central metabolism of Methylobacterium extorquens AM1. The flux rates predicted by the flux-minimization method exhibit significant correlations with flux rates obtained by either kinetic modelling or direct experimental determination. Larger deviations occur for segments of the network composed of redundant branches where the flux-minimization method always attributes the total flux to the thermodynamically most favourable branch. Nevertheless, compared with existing methods of structural modelling, the principle of flux minimization appears to be a promising theoretical approach to assess stationary flux rates in metabolic systems in cases where a detailed kinetic model is not yet available.

Programmed cell deaths

Abstract: Programmed cell death is a major component of both normal development and disease. The roles of cell death during either embryogenesis or pathogenesis, the signals that modulate this event, and the mechanisms of cell demise are the major subjects that drive research in this field. Increasing evidence obtained both in vitro and in vivo supports the hypothesis that a variety of cell death programs may be triggered in distinct circumstances. Contrary to the view that caspase-mediated apoptosis represents the standard programmed cell death, recent studies indicate that an apoptotic morphology can be produced independent of caspases, that autophagic execution pathways of cell death may be engaged without either the involvement of caspases or morphological signs of apoptosis, and that even the necrotic morphology of cell death may be consistently produced in some cases, including certain plants. Alternative cell death programs may imply novel therapeutic targets, with important consequences for attempts to treat diseases associated with disregulated programmed cell death.

Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

Abstract: We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.

Acyl-CoA dehydrogenases - a mechanistic overview

Abstract: Acyl-CoA dehydrogenases constitute a family of flavoproteins that catalyze the α,β-dehydrogenation of fatty acid acyl-CoA conjugates While they differ widely in their specificity, they share the same basic chemical mechanism of α,β-dehydrogenation Medium chain acyl-CoA dehydrogenase is probably the best-studied member of the class and serves as a model for the study of catalytic mechanisms Based on medium chain acyl-CoA dehydrogenase we discuss the main factors that bring about catalysis, promote specificity and determine the selective transfer of electrons to electron transferring flavoprotein The mechanism of α,β-dehydrogenation is viewed as a process in which the substrate αC-H and βC-H bonds are ruptured concertedly, the first hydrogen being removed by the active center base Glu376-COO– as an H+, the second being transferred as a hydride to the flavin N(5) position Hereby the pKa of the substrate αC-H is lowered from > 20 to ≈ 8 by the effect of specific hydrogen bonds Concomitantly, the pKa of Glu376-COO– is also raised to 8–9 due to the decrease in polarity brought about by substrate binding The kinetic sequence of medium chain acyl-CoA dehydrogenase is rather complex and involves several intermediates A prominent one is the molecular complex of reduced enzyme with the enoyl-CoA product that is characterized by an intense charge transfer absorption and serves as the point of transfer of electrons to the electron transferring flavoprotein These views are also discussed in the context of the accompanying paper on the three-dimensional properties of acyl-CoA dehydrogenases

Role of extracellular signal regulated kinases 1 and 2 in neuronal survival

Abstract: Extracellular signal regulated kinases 1 and 2 (ERK1/2) regulate cellular responses to a variety of extracellular stimuli. In the nervous system, ERK1/2 is critical for neuronal differentiation, plasticity and may also modulate neuronal survival. In this minireview, we present evidence that supports prosurvival activity of ERK1/2 in neurons. Several reports suggest that ERK1/2 mediates neuroprotective activity of extracellular factors, including neurotrophins. In addition, ERK1/2 is activated by neuronal injury. In damaged cells, ERK1/2 activation may act as a defensive mechanism that helps to compensate for the deleterious effects of a damaging insult. The emerging mechanisms of ERK1/2-mediated neuroprotection may involve transcriptional regulation and/or direct inhibition of cell death machinery.

Oxidative neuronal injury: The dark side of ERK1/2

Abstract: The extracellular signal regulated protein kinases (ERK1/2) are essential for normal development and functional plasticity of the central nervous system. However, a growing number of recent studies in models of cerebral ischemia, brain trauma and neurodegenerative diseases implicate a detrimental role for ERK1/2 signaling during oxidative neuronal injury. Neurons undergoing oxidative stress-related injuries typically display a biphasic or sustained pattern of ERK1/2 activation. A variety of potential targets of reactive oxygen species and reactive nitrogen species could contribute to ERK1/2 activation. These include cell surface receptors, G proteins, upstream kinases, protein phosphatases and proteasome components, each of which could be direct or indirect targets of reactive oxygen or nitrogen species, thereby modulating the duration and magnitude of ERK1/2 activation. Neuronal oxidative stress also appears to influence the subcellular trafficking and/or localization of activated ERK1/2. Differences in compartmentalization of phosphorylated ERK1/2 have been observed in diseased or injured human neurons and in their respective animal and cell culture model systems. We propose that differential accessibility of ERK1/2 to downstream targets, which is dictated by the persistent activation of ERK1/2 within distinct subcellular compartments, underlies the neurotoxic responses that are driven by this kinase.

Chromatin dynamics at DNA replication, transcription and repair

Abstract: During DNA replication, transcription and DNA repair in eukaryotes, the cellular machineries performing these tasks need to gain access to the DNA that is packaged into chromatin in the nucleus. Chromatin is a dynamic structure that modulates the access of regulatory factors to the genetic material. A precise coordination and organization of events in opening and closing of the chromatin is crucial to ensure that the correct spatial and temporal epigenetic code is maintained within the eukaryotic genome. This review will summarize the current knowledge of how chromatin remodeling and histone modifying complexes cooperate to break and remake chromatin during nuclear processes on the DNA template.

High level cell-free expression and specific labeling of integral membrane proteins

Abstract: We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein ‘remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced 1 by formation of the proposed a-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15 N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.

The role of histones in chromatin remodelling during mammalian spermiogenesis.

Abstract: One of the most dramatic chromatin remodelling processes takes place during mammalian spermatogenesis. Indeed, during the postmeiotic maturation of male haploid germ cells, or spermiogenesis, histones are replaced by small basic proteins, which in mammals are transition proteins and protamines. However, nothing is known of the mechanisms controlling the process of histone replacement. Two hints from the literature could help to shed light on the underlying molecular events: one is the massive synthesis of histone variants, including testis-specific members, and the second is a stage specific post-translational modification of histones. A new testis-specific 'histone code' can therefore be generated combining both histone variants and histone post-translational modifications. This review will detail these two phenomena and discuss possible functional significance of the global chromatin alterations occurring prior to histone replacement during spermiogenesis.

Structure and function of plant aspartic proteinases.

Abstract: Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana ,n ew questions concerning novel structure–function relationships among plant aspartic proteinases are now starting to be

A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

Abstract: Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells.

Abstract: Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf-binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf-binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium-affinity interactions ( approximately 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface-expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti-human immunodeficiency virus pseudopeptide, HB-19, which binds specifically surface-expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB-19 and various Lf derivatives in proteoglycan-deficient hamster cells suggested that the nucleolin-binding site is located in both the N- and C-terminal lobes of Lf, whereas the basic N-terminal region is dispensable. On intact cells, Lf co-localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB-19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan-deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.

Enzymes in organic media. Forms, functions and applications.

Abstract: Enzyme catalysis in low water containing organic solvents is finding an increasing number of applications in diverse areas. This review focuses on some aspects which have not been reviewed elsewhere. Different strategies for obtaining higher activity and stability in such media are described. In this context, the damaging role of lyophilization and the means of overcoming such effects are discussed. Ultrasonication and microwave assistance are two emerging approaches for enhancing reaction rates in low water media. Control of water activity and medium engineering are two crucial approaches in optimization of catalytic behaviour in nonaqueous enzymology. Organometallics and synthesis/modification of polymers are two areas where nonaqueous enzymology can play a greater role in the coming years. The greater understanding of enzyme behaviour in nonaqueous media is expected to lead to larger and even more diverse kinds of applications.

Collectins: players of the innate immune system.

Abstract: Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

Antimicrobial activities of heparin-binding peptides

Abstract: Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, and fibronectin, exerted antimicrobial activities against Gram-positive and Gram-negative bacteria. Similar results were obtained using heparin-binding peptides derived from complement factor C3 as well as consensus sequences for heparin-binding (Cardin and Weintraub motifs). These sequence motifs, and additional peptides, also killed the fungus Candida albicans. These data will have implications for the search for novel antimicrobial peptides and utilization of heparin-protein interactions should be helpful in the identification and purification of novel antimicrobial peptides from complex biological mixtures. Finally, consensus regions may serve as templates for de novo synthesis of novel antimicrobial molecules.

Genetic approaches to the cellular functions of polyamines in mammals

Abstract: The polyamines putrescine, spermidine and spermine are organic cations shown to participate in a bewildering number of cellular reactions, yet their exact functions in intermediary metabolism and specific interactions with cellular components remain largely elusive Pharmacological interventions have demonstrated convincingly that a steady supply of these compounds is a prerequisite for cell proliferation to occur The last decade has witnessed the appearance of a substantial number of studies, in which genetic engineering of polyamine metabolism in transgenic rodents has been employed to unravel their cellular functions Transgenic activation of polyamine biosynthesis through an overexpression of their biosynthetic enzymes has assigned specific roles for these compounds in spermatogenesis, skin physiology, promotion of tumorigenesis and organ hypertrophy as well as neuronal protection Transgenic activation of polyamine catabolism not only profoundly disturbs polyamine homeostasis in most tissues, but also creates a complex phenotype affecting skin, female fertility, fat depots, pancreatic integrity and regenerative growth Transgenic expression of ornithine decarboxylase antizyme has suggested that this unique protein may act as a general tumor suppressor Homozygous deficiency of the key biosynthetic enzymes of the polyamines, ornithine and S-adenosylmethionine decarboxylase, as achieved through targeted disruption of their genes, is not compatible with murine embryogenesis Finally, the first reports of human diseases apparently caused by mutations or rearrangements of the genes involved in polyamine metabolism have appeared

UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea The enzyme contains functional iron after all

Abstract: H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed ‘metal-free’ hydrogenase does not contain iron–sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron–sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does, however, harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A (320–400 nm) or blue-light (400–500 nm). Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A/blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV/visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark, Hmd consistently contained per monomer about one Fe, which was tightly bound to the cofactor. The iron was released from the enzyme and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as ‘metal-free’ hydrogenase is no longer appropriate.

Unraveling DNA helicases. Motif, structure, mechanism and function.

Abstract: DNA helicases are molecular 'motor' enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.

Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase.

Abstract: Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.

Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences.

Abstract: Acyl-CoA dehydrogenases and acyl-CoA oxidases are two closely related FAD-containing enzyme families that are present in mitochondria and peroxisomes, respectively. They catalyze the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. This review examines the structure of medium chain acyl-CoA dehydrogenase, as a representative of the dehydrogenase family, with respect to the catalytic mechanism and its broad chain length specificity. Comparing the structures of four other acyl-CoA dehydrogenases provides further insights into the structural basis for the substrate specificity of each of these enzymes. In addition, the structure of peroxisomal acyl-CoA oxidase II from rat liver is compared to that of medium chain acyl-CoA dehydrogenase, and the structural basis for their different oxidative half reactions is discussed.

Principles behind the multifarious control of signal transduction. ERK phosphorylation and kinase/phosphatase control.

Abstract: General and simple principles are identified that govern signal transduction. The effects of kinase and phosphatase inhibition on a MAP kinase pathway are first examined in silica. Quantitative measures for the control of signal amplitude, duration and integral strength are introduced. We then identify and prove new principles, such that total control on signal amplitude and on final signal strength must amount to zero, and total control on signal duration and on integral signal intensity must equal -1. Collectively, kinases control amplitudes more than duration, whereas phosphatases tend to control both. We illustrate and validate these principles experimentally: (a) a kinase inhibitor affects the amplitude of EGF-induced ERK phosphorylation much more than its duration and (b) a phosphatase inhibitor influences both signal duration and signal amplitude, in particular long after EGF administration. Implications for the cellular decision between growth and differentiation are discussed.

The optimization of protein secondary structure determination with infrared and circular dichroism spectra

Abstract: We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.

The PAS fold. A redefinition of the PAS domain based upon structural prediction.

Abstract: In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the problems with the current annotation of the PAS domain is that this domain exhibits limited similarity at the amino acid sequence level. It is therefore essential, when using proteins with low-sequence similarities, to apply profile hidden Markov model searches for the PAS domain-containing proteins, as for the PFAM database. From recent 3D X-ray and NMR structures, however, PAS domains appear to have a conserved 3D fold as shown here by structural alignment of the six representative 3D-structures from the PDB database. Large-scale modelling of the PAS sequences from the PFAM database against the 3D-structures of these six structural prototypes was performed. All 3D models generated (> 5700) were evaluated using prosaii. We conclude from our large-scale modelling studies that the PAS and PAC motifs (which are separately defined in the PFAM database) are directly linked and that these two motifs form the PAS fold. The existing subdivision in PAS and PAC motifs, as used by the PFAM and SMART databases, appears to be caused by major differences in sequences in the region connecting these two motifs. This region, as has been shown by Gardner and coworkers for human PAS kinase (Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. (2002) Structure 10, 1349-1361, [1]), is very flexible and adopts different conformations depending on the bound ligand. Some PAS sequences present in the PFAM database did not produce a good structural model, even after realignment using a structure-based alignment method, suggesting that these representatives are unlikely to have a fold resembling any of the structural prototypes of the PAS domain superfamily.

On the mechanism of action of the antifungal agent propionate.

Abstract: Propionate is used to protect bread and animal feed from moulds. The mode of action of this short-chain fatty acid was studied using Aspergillus nidulans as a model organism. The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl-CoA via propionyl-CoA, methylcitrate and pyruvate. Propionate inhibits growth of A. nidulans on glucose but not on acetate; the latter was shown to inhibit propionate oxidation. When grown on glucose a methylcitrate synthase deletion mutant is much more sensitive towards the presence of propionate in the medium as compared to the wild-type and accumulates 10-fold higher levels of propionyl-CoA, which inhibits CoA-dependent enzymes such as pyruvate dehydrogenase, succinyl-CoA synthetase and ATP citrate lyase. The most important inhibition is that of pyruvate dehydrogenase, as this affects glucose and propionate metabolism directly. In contrast, the blocked succinyl-CoA synthetase can be circumvented by a succinyl-CoA:acetate/propionate CoA-transferase, whereas ATP citrate lyase is required only for biosynthetic purposes. In addition, data are presented that correlate inhibition of fungal polyketide synthesis by propionyl-CoA with the accumulation of this CoA-derivative. A possible toxicity of propionyl-CoA for humans in diseases such as propionic acidaemia and methylmalonic aciduria is also discussed.

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp

Abstract: The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µm while a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.

Weapons of STAT destruction

Abstract: The signal transducer and activator of transcription (STAT) family of proteins function to activate gene transcription downstream of myriad cytokine and growth factor signals. The prototype STAT proteins, STAT1 and STAT2, are required for innate and adaptive antimicrobial immune responses that result from interferon signal transduction. While many viruses have evolved the ability to avoid these antiviral cytokines, the Paramyxoviruses are distinct in their abilities to interfere directly with STAT proteins. Individual paramyxovirus species differ greatly in their precise mechanism of STAT signaling evasion, but a virus-encoded protein called V plays a central role in this process. The theme of V-dependent interferon evasion and its variations provide significant insights into virus-host interactions and viral immune evasion that can help define targets for antiviral drug design. Exposure of the viral weapons of STAT destruction may also be instructive for application to STAT-directed therapeutics for diseases characterized by STAT hyperactivity.