Showing papers in "FEBS Journal in 2007"

ER stress and diseases

Abstract: Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells activate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and translational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorder, which are collectively known as 'conformational diseases'. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases.

Energy metabolism in tumor cells

Abstract: In early studies on energy metabolism of tumor cells, it was proposed that the enhanced glycolysis was induced by a decreased oxidative phosphorylation. Since then it has been indiscriminately applied to all types of tumor cells that the ATP supply is mainly or only provided by glycolysis, without an appropriate experimental evaluation. In this review, the different genetic and biochemical mechanisms by which tumor cells achieve an enhanced glycolytic flux are analyzed. Furthermore, the proposed mechanisms that arguably lead to a decreased oxidative phosphorylation in tumor cells are discussed. As the O(2) concentration in hypoxic regions of tumors seems not to be limiting for the functioning of oxidative phosphorylation, this pathway is re-evaluated regarding oxidizable substrate utilization and its contribution to ATP supply versus glycolysis. In the tumor cell lines where the oxidative metabolism prevails over the glycolytic metabolism for ATP supply, the flux control distribution of both pathways is described. The effect of glycolytic and mitochondrial drugs on tumor energy metabolism and cellular proliferation is described and discussed. Similarly, the energy metabolic changes associated with inherent and acquired resistance to radiotherapy and chemotherapy of tumor cells, and those determined by positron emission tomography, are revised. It is proposed that energy metabolism may be an alternative therapeutic target for both hypoxic (glycolytic) and oxidative tumors.

Vitamin C. Biosynthesis, recycling and degradation in mammals.

Abstract: Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.

Muscle and neuronal nicotinic acetylcholine receptors

Abstract: Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon) There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc) Extensive research has determined the specific function of several nAChR subtypes nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology

Metabonomics in pharmaceutical R&D.

Abstract: This minireview is based on a lecture given at the First Maga Circe Conference on metabolomics held at Sabaudia, Italy, in March 2006 in which the analytical and statistical techniques used in metabonomics, efforts at standardization and some of the major applications to pharmaceutical research and development are reviewed. Metabonomics involves the determination of multiple metabolites simultaneously in biofluids, tissues and tissue extracts. Applications to preclinical drug safety studies are illustrated by the Consortium for Metabonomic Toxicology, a collaboration involving several major pharmaceutical companies. This consortium was able, through the measurement of a dataset of NMR spectra of rodent urine and serum samples, to build a predictive expert system for liver and kidney toxicity. A secondary benefit was the elucidation of the endogenous biochemicals responsible for the classification. The use of metabonomics in disease diagnosis and therapy monitoring is discussed with an exemplification from coronary artery disease, and the concept of pharmaco-metabonomics as a way of predicting an individual's response to treatment is exemplified. Finally, some advantages and perceived difficulties of the metabonomics approach are summarized.

Malaria - an overview.

Abstract: Malaria is caused by protozoan parasites of the genus Plasmodium and is a major cause of mortality and morbidity worldwide. These parasites have a complex life cycle in their mosquito vector and vertebrate hosts. The primary factors contributing to the resurgence of malaria are the appearance of drug-resistant strains of the parasite, the spread of insecticide-resistant strains of the mosquito and the lack of licensed malaria vaccines of proven efficacy. This minireview includes a summary of the disease, the life cycle of the parasite, information relating to the genome and proteome of the species lethal to humans, Plasmodium falciparum, together with other recent developments in the field.

Fermentative lifestyle in yeasts belonging to the Saccharomyces complex

Abstract: The yeast Saccharomyces cerevisiae is characterized by its ability to: (a) degrade glucose or fructose to ethanol, even in the presence of oxygen (Crabtree effect); (b) grow in the absence of oxygen; and (c) generate respiratory-deficient mitochondrial mutants, so-called petites. How unique are these properties among yeasts in the Saccharomyces clade, and what is their origin? Recent progress in genome sequencing has elucidated the phylogenetic relationships among yeasts in the Saccharomyces complex, providing a framework for the understanding of the evolutionary history of several modern traits. In this study, we analyzed over 40 yeasts that reflect over 150 million years of evolutionary history for their ability to ferment, grow in the absence of oxygen, and generate petites. A great majority of isolates exhibited good fermentation ability, suggesting that this trait could already be an intrinsic property of the progenitor yeast. We found that lineages that underwent the whole-genome duplication, in general, exhibit a fermentative lifestyle, the Crabtree effect, and the ability to grow without oxygen, and can generate stable petite mutants. Some of the pre-genome duplication lineages also exhibit some of these traits, but a majority of the tested species are petite-negative, and show a reduced Crabtree effect and a reduced ability to grow in the absence of oxygen. It could be that the ability to accumulate ethanol in the presence of oxygen, a gradual independence from oxygen and/or the ability to generate petites were developed later in several lineages. However, these traits have been combined and developed to perfection only in the lineage that underwent the whole-genome duplication and led to the modern Saccharomyces cerevisiae yeast.

Protein lipidation: Function and mechanism of palmitoylation

Abstract: Proteins are covalently modified with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins. The focus of this review is S‐palmitoylation, the reversible addition of palmitate and other long‐chain fatty acids to proteins at cysteine residues in a variety of sequence contexts. The functional consequences of palmitoylation are diverse. Palmitoylation facilitates the association of proteins with membranes, mediates protein trafficking, and more recently has been appreciated as a regulator of protein stability. Members of a family of integral membrane proteins that harbor a DHHC cysteine‐rich domain mediate most cellular palmitoylation events. Here we focus on DHHC proteins that modify Ras proteins in yeast and mammalian cells.

High-resolution NMR studies of the zinc-binding site of the Alzheimer's amyloid β-peptide

Abstract: Metal binding to the amyloid β-peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high-resolution NMR to study zinc binding to amyloid β-peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo-form and the holo-form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N-relaxation and amide proton exchange experiments on amyloid β-peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N-terminus, similar to a previously proposed copper-binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β-peptide. The dissociation constant Kd of zinc for the fragment amyloid β-peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β-peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal-induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal-induced structure of the peptide counteracts aggregation.

Drug-resistant malaria - an insight.

Abstract: Despite intensive research extending back to the 1930s, when the first synthetic antimalarial drugs made their appearance, the repertoire of clinically licensed formulations remains very limited. Moreover, widespread and increasing resistance to these drugs contributes enormously to the difficulties in controlling malaria, posing considerable intellectual, technical and humanitarian challenges. A detailed understanding of the molecular mechanisms underlying resistance to these agents is emerging that should permit new drugs to be rationally developed and older ones to be engineered to regain their efficacy. This review summarizes recent progress in analysing the causes of resistance to the major antimalarial drugs and its spread.

Nanoparticle-enzyme hybrid systems for nanobiotechnology.

Abstract: Biomolecule–nanoparticle (NP) [or quantum-dot (QD)] hybrid systems combine the recognition and biocatalytic properties of biomolecules with the unique electronic, optical, and catalytic features of NPs and yield composite materials with new functionalities. The biomolecule–NP hybrid systems allow the development of new biosensors, the synthesis of metallic nanowires, and the fabrication of nanostructured patterns of metallic or magnetic NPs on surfaces. These advances in nanobiotechnology are exemplified by the development of amperometric glucose sensors by the electrical contacting of redox enzymes by means of AuNPs, and the design of an optical glucose sensor by the biocatalytic growth of AuNPs. The biocatalytic growth of metallic NPs is used to fabricate Au and Ag nanowires on surfaces. The fluorescence properties of semiconductor QDs are used to develop competitive maltose biosensors and to probe the biocatalytic functions of proteases. Similarly, semiconductor NPs, associated with electrodes, are used to photoactivate bioelectrocatalytic cascades while generating photocurrents.

Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers.

Abstract: Glutathione peroxidases (GPXs, EC 1.11.1.9) were first discovered in mammals as key enzymes involved in scavenging of activated oxygen species (AOS). Their efficient antioxidant activity depends on the presence of the rare amino-acid residue selenocysteine (SeCys) at the catalytic site. Nonselenium GPX-like proteins (NS-GPXs) with a Cys residue instead of SeCys have also been found in most organisms. As SeCys is important for GPX activity, the function of the NS-GPX can be questioned. Here, we highlight the evolutionary link between NS-GPX and seleno-GPX, particularly the evolution of the SeCys incorporation system. We then discuss what is known about the enzymatic activity and physiological functions of NS-GPX. Biochemical studies have shown that NS-GPXs are not true GPXs; notably they reduce AOS using reducing substrates other than glutathione, such as thioredoxin. We provide evidence that, in addition to their inefficient scavenging action, NS-GPXs act as AOS sensors in various signal-transduction pathways.

CREB and cAMP response element‐mediated gene expression in the ischemic brain

Abstract: Cerebral ischemia triggers robust phosphorylation of cAMP response element-binding protein (CREB) and CRE-mediated gene expression in neurons. Glutamate receptor activation and subsequent calcium influx may activate CREB shortly after ischemia. CREB activation leads to expression of genes encoding neuroprotective molecules, such as the antiapoptotic protein Bcl-2, and contributes to survival of neurons after ischemic insult. Recent studies have suggested that CREB may be involved in acquisition of ischemic tolerance, a phenomenon that occurs after sublethal ischemic stress. CREB activation is also involved in the survival of newborn neurons in the dentate gyrus of the hippocampus after ischemia. Therefore, CREB-related therapeutics may be promising for brain protection and endogenous neurogenesis and could promote functional recovery in ischemic stroke patients. This minireview summarizes our current understanding for the role of CREB in regulating CRE-mediated gene expression during cerebral ischemia.

Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics.

Abstract: For the characterization of protein sequences and post-translational modifications by MS, the 'top-down' proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used 'bottom-up' approach, which instead uses such data of peptides from the protein's digestion, but the top-down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False-positive rates for the identification of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient. Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top-down approach has a approximately 500 residue, approximately 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas-phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray ('prefolding dissociation'). This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).

Biomechanical properties of native basement membranes.

Abstract: Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Hyperthermophilic enzymes--stability, activity and implementation strategies for high temperature applications.

Abstract: Current theories agree that there appears to be no unique feature responsible for the remarkable heat stability properties of hyperthermostable proteins. A concerted action of structural, dynamic and other physicochemical attributes are utilized to ensure the delicate balance between stability and functionality of proteins at high temperatures. We have thoroughly screened the literature for hyperthermostable enzymes with optimal temperatures exceeding 100 degrees C that can potentially be employed in multiple biotechnological and industrial applications and to substitute traditionally used, high-cost engineered mesophilic/thermophilic enzymes that operate at lower temperatures. Furthermore, we discuss general methods of enzyme immobilization and suggest specific strategies to improve thermal stability, activity and durability of hyperthermophilic enzymes.

Osmotic stress sensing and signaling in fishes.

Abstract: In their aqueous habitats, fish are exposed to a wide range of osmotic conditions and differ in their abilities to respond adaptively to these variations in salinity. Fish species that inhabit environments characterized by significant salinity fluctuation (intertidal zone, estuaries, salt lakes, etc.) are euryhaline and able to adapt to osmotic stress. Adaptive and acclimatory responses of fish to salinity stress are based on efficient mechanisms of osmosensing and osmotic stress signaling. Multiple osmosensors, including calcium sensing receptor likely act in concert to convey information about osmolality changes to downstream signaling and effector mechanisms. The osmosensory signal transduction network in fishes is complex and includes calcium, mitogen-activated protein kinase, 14-3-3 and macromolecular damage activated signaling pathways. This network controls, among other targets, osmosensitive transcription factors such as tonicity response element binding protein and osmotic stress transcription factor 1, which, in turn, regulate the expression of genes involved in osmotic stress acclimation. In addition to intracellular signaling mechanisms, the systemic response to osmotic stress in euryhaline fish is coordinated via hormone- and paracrine factor-mediated extracellular signaling. Overall, current insight into osmosensing and osmotic stress-induced signal transduction in fishes is limited. However, euryhaline fish species represent excellent models for answering critical emerging questions in this field and for elucidating the underlying molecular mechanisms of osmosensory signal transduction.

S-Layers as a basic building block in a molecular construction kit.

Abstract: Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.

Transthyretin and familial amyloidotic polyneuropathy. Recent progress in understanding the molecular mechanism of neurodegeneration.

Abstract: Familial amyloidotic polyneuropathy (FAP) is an inherited autosomal dominant disease that is commonly caused by accumulation of deposits of transthyretin (TTR) amyloid around peripheral nerves. The only effective treatment for FAP is liver transplantation. However, recent studies on TTR aggregation provide clues to the mechanism of the molecular pathogenesis of FAP and suggest new avenues for therapeutic intervention. It is increasingly recognized that there are common features of a number of protein-misfolding diseases that can lead to neurodegeneration. As for other amyloidogenic proteins, the most toxic forms of aggregated TTR are likely to be the low-molecular-mass diffusible species, and there is increasing evidence that this toxicity is mediated by disturbances in calcium homeostasis. This article reviews what is already known about the mechanism of TTR aggregation in FAP and describes how recent discoveries in other areas of amyloid research, particularly Alzheimer's disease, provide clues to the molecular pathogenesis of FAP.

Regulation of output from the plant circadian clock

Abstract: Plants, like many other organisms, have endogenous biological clocks that enable them to organize their physiological, metabolic and developmental processes so that they occur at optimal times. The best studied of these biological clocks are the circadian systems that regulate daily (approximately 24 h) rhythms. At the core of the circadian system in every organism are oscillators responsible for generating circadian rhythms. These oscillators can be entrained (set) by cues from the environment, such as daily changes in light and temperature. Completing the circadian clock model are the output pathways that provide a link between the oscillator and the various biological processes whose rhythms it controls. Over the past few years there has been a tremendous increase in our understanding of the mechanisms of the oscillator and entrainment pathways in plants and many useful reviews on the subject. In this review we focus on the output pathways by which the oscillator regulates rhythmic plant processes. In the first part of the review we describe the role of the circadian system in regulation at all stages of a plant's development, from germination and growth to reproductive development as well as in multiple cellular processes. Indeed, the importance of a circadian clock for plants can be gauged by the fact that so many facets of plant development are under its control. In the second part of the review we describe what is known about the mechanisms by which the circadian system regulates these output processes.

Recent applications of NMR spectroscopy in plant metabolomics.

Abstract: Recent research has established NMR as a key method for high-throughput comparative analysis of plant extracts. We discuss recent examples of the use of NMR to provide metabolomic data for various applications in plant science and look forward to the key role that NMR will play in data provision for plant systems biology.

Tumor suppressor p16INK4a--modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells.

Abstract: Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.

Substrate recognition by the protein disulfide isomerases

Abstract: Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate-limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol–disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate-binding sites and on determining their natural substrates in vivo.

Dynamics of α‐synuclein aggregation and inhibition of pore‐like oligomer development by β‐synuclein

Abstract: Accumulation of alpha-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, beta-synuclein (beta-syn), a close homologue, does not aggregate and reduces alpha-synuclein (alpha-syn)-related pathology. Although considerable information is available about the conformation of alpha-syn at the initial and end stages of fibrillation, less is known about the dynamic process of alpha-syn conversion to oligomers and how interactions with antiaggregation chaperones such as beta-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of alpha-syn showed that alpha-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating alpha-syn dimers on the membrane incorporate additional alpha-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, beta-syn dimers do not propagate and block the aggregation of alpha-syn into ring-like oligomers. Under in vitro cell-free conditions, alpha-syn aggregates formed ring-like structures that were disrupted by beta-syn. Similarly, cells expressing alpha-syn displayed increased ion current activity consistent with the formation of Zn(2+)-sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, alpha-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of beta-synuclein might be related to its ability to block the formation of pore-like structures.

CREB − a real culprit in oncogenesis

Abstract: The cAMP response element-binding protein (CREB) is a stimulus-induced transcription factor that responds rapidly to phosphorylation and/or coactivator activation. Regulated activation of CREB has a significant impact on cellular growth, proliferation and survival. To overturn the cellular control of these processes, tumor cells have developed various mechanisms to achieve constitutive activation of CREB, including gene amplification, chromosome translocation, interaction with viral oncoproteins, and inactivation of tumor suppressor genes. These mechanisms converge on the phosphorylation of CREB and/or the activation of transducer of regulated CREB activity (TORC) coactivators to effect uncontrolled proliferation of cells. This minireview summarizes the different lines of existing evidence that support a direct role of CREB in oncogenesis.

The glycosaminoglycan chain of decorin plays an important role in collagen fibril formation at the early stages of fibrillogenesis

Abstract: Decorin is a multifunctional small leucine-rich proteoglycan involved in the regulation of collagen fibrillogenesis. In patients with a variant of Ehlers-Danlos syndrome, about half of the secreted decorin lacks the single glycosaminoglycan side chain. Notably, these patients have a skin-fragility phenotype that resembles that of decorin null mice. In this study, we investigated the role of glycanated and unglycanated decorin on collagen fibrillogenesis. Glycosaminoglycan-free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN-S4A), showed reduced inhibition of fibrillogenesis compared with the decorin proteoglycan. Interestingly, using a 3D matrix generated by decorin-null fibroblasts, an increase in fibril diameter was found after the addition of decorin, and even greater effects were observed with DCN-S4A. To avoid potential side effects of artificial tags, adenoviruses containing decorin and DCN-S4A were used to transduce decorin-null fibroblasts prior to matrix formation. Both molecules were efficiently incorporated into the matrix, with no changes in collagen composition and network formation, or altered expression of the related proteoglycan biglycan. Both decorin and DCN-S4A mutants increased the collagen fibril diameter, with the latter showing the most prominent effects. These data show that at early stages of fibrillogenesis, the glycosaminoglycan chain of decorin has a reducing effect on collagen fibril diameter.

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice.

Abstract: The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.

Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase

Abstract: The crystalline polymorphic form of cellulose (cellulose Iα-rich) of the green alga, Cladophora, was converted into cellulose IIII and Iβ by supercritical ammonium and hydrothermal treatments, respectively, and the hydrolytic rate and the adsorption of Trichoderma viride cellobiohydrolase I (Cel7A) on these products were evaluated by a novel analysis based on the surface density of the enzyme. Cellobiose production from cellulose IIII was more than 5 times higher than that from cellulose I. However, the amount of enzyme adsorbed on cellulose IIII was less than twice that on cellulose I, and the specific activity of the adsorbed enzyme for cellulose IIII was more than 3 times higher than that for cellulose I. When cellulose IIII was converted into cellulose Iβ by hydrothermal treatment, cellobiose production was dramatically decreased, although no significant change was observed in enzyme adsorption. This clearly indicates that the enhanced hydrolysis of cellulose IIII is related to the structure of the crystalline polymorph. Thus, supercritical ammonium treatment activates crystalline cellulose for hydrolysis by cellobiohydrolase.

The nuclear lamina. Both a structural framework and a platform for genome organization.

Abstract: The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one-layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson–Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the ‘behaviour’ of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina-associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.