Showing papers in "Biochemistry in 2009"
TL;DR: An introduction to the impact of click chemistry and other bioorthogonal reactions on the study of biological systems is presented and includes discussion of the philosophy behind click chemistry, the details and benefits of bioorthogsonal reactions that have been developed, and examples of recent innovative approaches that have effectively exploited these transformations.
Abstract: In recent years, a number of bioorthogonal reactions have been developed, exemplified by click chemistry, that enable the efficient formation of a specific product, even within a highly complex chemical environment. While the exquisite selectivity and reliability of these transformations have led to their broad application in diverse research areas, they have proven to be particularly beneficial to biological studies. In this regard, the ability to rationally incorporate reactive tags onto a biomolecular target and subsequently achieve high selectivity in tag derivatization within a complex biological sample has revolutionized the toolbox that is available for addressing fundamental issues. Herein, an introduction to the impact of click chemistry and other bioorthogonal reactions on the study of biological systems is presented. This includes discussion of the philosophy behind click chemistry, the details and benefits of bioorthogonal reactions that have been developed, and examples of recent innovative approaches that have effectively exploited these transformations to study cellular processes. For the latter, the impacts of bioorthogonal reactions on drug design (i.e., in situ combinatorial drug design), biomolecule labeling and detection (site-specific derivatization of proteins, viruses, sugars, DNA, RNA, and lipids), and the recent strategy of activity-based protein profiling are highlighted.
617 citations
TL;DR: It is proposed that Glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol.
Abstract: The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.
420 citations
TL;DR: Using NMR, the NMR data indicate that soluble oligomers of Abeta preglobulomer and globulomer have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets.
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid β-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer’s patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid β-peptides. Using NMR, we have characterized soluble oligomers of Aβ preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel β-sheet structure that is different from fibrils which contain only parallel β-sheets. Using the structural data, we engineered a disulfide bond into the soluble Aβ globulomer to give a “new” soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.
347 citations
TL;DR: A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution, and features include a novel interface with an antiparallel arrangement of beta chains and a unique tangential association of coiled coils from neighboring molecules.
Abstract: A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution. The protein was purified from human blood plasma, first by a cold ethanol precipitation procedure and then by stepwise chromatography on DEAE-cellulose. A product was obtained that was homogeneous on SDS−polyacrylamide gels. Nonetheless, when individual crystals used for X-ray diffraction were examined by SDS gel electrophoresis after data collection, two species of α chain were present, indicating that some proteolysis had occurred during the course of operations. Amino-terminal sequencing on post-X-ray crystals showed mostly intact native α- and γ-chain sequences (the native β chain is blocked). The overall structure differs from that of a native fibrinogen from chicken blood and those reported for a partially proteolyzed bovine fibrinogen in the nature of twist in the coiled-coil regions, likely due to weak forces imparted by unique crystal packing. As such, the structure adds to the inventory of possible con...
312 citations
TL;DR: The unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton is reported and it is suggested that Pink1 functions in the trafficking of mitochondria in cells.
Abstract: Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease. Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. Here, we report the unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. Our screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, we postulate here a role for Pink1 in mitochondrial trafficking. Using subcellular fractionation, we show that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, we document that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, we find that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. Our findings suggest that Pink1 functions in the trafficking of mitochondria in cells.
287 citations
TL;DR: This review summarizes the current structural and mechanistic information available that can be utilized in the development of future highly specific neuroprotectants and cardioprotectants.
Abstract: The past decade has brought major advances in our knowledge of the structures and mechanisms of MAO A and MAO B, which are pharmacological targets for specific inhibitors. In both enzymes, crystallographic and biochemical data show their respective C-terminal transmembrane helices anchor the enzymes to the outer mitochondrial membrane. Pulsed EPR data show both enzymes are dimeric in their membrane-bound forms with agreement between distances measured in their crystalline forms. Distances measured between active site-directed spin-labels in membrane preparations show excellent agreement with those estimated from crystallographic data. Our knowledge of requirements for development of specific reversible MAO B inhibitors is in a fairly mature status. Less is known regarding the structural requirements for highly specific reversible MAO A inhibitors. In spite of their 70% level of sequence identity and similarities of Cα folds, the two enzymes exhibit significant functional and structural differences that ca...
265 citations
TL;DR: The hypothesis that it is the thermodynamics of the insertion of the peptide into the membrane, from a surface-bound state, that determine the mechanism is proposed.
Abstract: The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies; however, they all form amphipathic alpha-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely, binding and peptide-induced dye efflux. The thermodynamics of binding calculated using the Wimley-White interfacial hydrophobicity scale are in good agreement with the values derived from experiment. The generally accepted view that binding affinity determines functional specificity is also supported by experiments in model membranes. We now propose the hypothesis that it is the thermodynamics of the insertion of the peptide into the membrane, from a surface-bound state, that determine the mechanism.
263 citations
TL;DR: The principle of DNA self-assembly is summarized, the exciting progress of structural DNA nanotechnology in recent years is described, and the current frontier is discussed.
Abstract: Naturally existing biological systems, from the simplest unicellular diatom to the most sophisticated organ such as the human brain, are functional self-assembled architectures. Scientists have long been dreaming about building artificial nanostructures that can mimic such elegance in nature. Structural DNA nanotechnology, which uses DNA as a blueprint and building material to organize matter with nanometer precision, represents an appealing solution to this challenge. On the basis of the knowledge of helical DNA structure and Watson-Crick base pairing rules, scientists have constructed a number of DNA nanoarchitectures with a large variety of geometries, topologies, and periodicities with considerably high yields. Modified by functional groups, those DNA nanostructures can serve as scaffolds to control the positioning of other molecular species, which opens opportunities to study intermolecular synergies, such as protein-protein interactions, as well as to build artificial multicomponent nanomachines. In this review, we summarize the principle of DNA self-assembly, describe the exciting progress of structural DNA nanotechnology in recent years, and discuss the current frontier.
248 citations
TL;DR: This review explores the structural basis of sHSP oligomeric flexibility and the corresponding functional consequences in the context of a model describing sH SP activity with a set of three coupled thermodynamic equilibria and concludes that such a conceptual framework provides a sound foundation for describing these emerging functions in physiological and pathological processes.
Abstract: Small heat shock proteins (sHSP) make up a remarkably diverse group of molecular chaperones possessing a degree of structural plasticity unparalleled in other protein superfamilies. In the absence of chemical energy input, these stability sensors can sensitively recognize and bind destabilized proteins, even in the absence of gross misfolding. Cellular conditions regulate affinity toward client proteins, allowing tightly controlled switching and tuning of sHSP chaperone capacity. Perturbations of this regulation, through chemical modification or mutation, directly lead to a variety of disease states. This review explores the structural basis of sHSP oligomeric flexibility and the corresponding functional consequences in the context of a model describing sHSP activity with a set of three coupled thermodynamic equilibria. As current research illuminates many novel physiological roles for sHSP outside of their traditional duties as molecular chaperones, such a conceptual framework provides a sound foundation for describing these emerging functions in physiological and pathological processes.
239 citations
TL;DR: The general peroxidase activity of G-quadruplex-hemin complexes is demonstrated to be applicable for screening G- quadruplex ligands (potential anticancer reagents) using colorimetric and visual detection strategies.
Abstract: DNA sequences with repetitive G-rich structural motifs, which form special structures called G-quadruplexes, widely exist in the human genome. Here we report the general peroxidase activity of G-quadruplex−hemin complexes and discuss the connection between peroxidase activity and G-quadruplex structures. The high peroxidase activity of hemin complexed with intramolecular parallel G-quadruplex-forming sequences in gene promoters (such as c-Myc, VEGF, c-Kit21, HIF-1α, and RET) may imply a potential mechanism of hemin-mediated cellular injury. This peroxidase activity has also been demonstrated to be applicable for screening G-quadruplex ligands (potential anticancer reagents) using colorimetric and visual detection strategies.
234 citations
TL;DR: It is shown that the MYB transcription factors play a key role in plant development, such as secondary metabolism, hormone signal transduction, disease resistance, cell shape, organ development, etc.
Abstract: MYB genes are widely distributed in higher plants and comprise one of the largest transcription factors, which are characterized by the presence of a highly conserved MYB domain at their N-termini. Over recent decades, biochemical and molecular characterizations of MYB have been extensively studied and reported to be involved in many physiological and biochemical processes. This review describes current knowledge of their structure characteristic, classification, multi-functionality, mechanism of combinational control, evolution, and function redundancy. It shows that the MYB transcription factors play a key role in plant development, such as secondary metabolism, hormone signal transduction, disease resistance, cell shape, organ development, etc. Furthermore, the expression of some members of the MYB family shows tissuespecificity.
TL;DR: The second edition of the book as mentioned in this paper is dedicated to the memory of Rosalind Kornfeld and Roger Jeanloz, pioneers in the elucidation of glycan structure and func tion.
Abstract: years ago (see for book review Biochemistry (Moscow), No. 8, 2000). Glycobiology has advanced significantly during the past decade, and publishing of the second edi tion of this book is very timely. The book is dedicated to the memory of Rosalind Kornfeld and Roger Jeanloz, pioneers in the elucidation of glycan structure and func tion. The book consists of 51 chapters, glossary, study guide, and subject index. The first part, “General Principles”, includes seven chapters that focus on the historical background of glyco biology and overview of major important subjects that form the of glycobiology: monosaccharides, major classes of glycoconjugates and glycans, their structure, biological role, diversity and site specific glycosylation. The sepa rate chapters of this part cover cellular organization of glycosylation, glycosyl transferases, and a genomic view of glycobiology. The second part highlights structure and biosynthe sis of glycans. There are data about structure of various types of N glycans, glycosphingolipids, proteoglycans and sulfated glycosaminoglycans, sialic acids, and hyaluronan. The third part is devoted to organismal diversity. The evolution of glycan diversity in bacteria, fungi, viridiplan tae, nematode, arthropoda, and deuterostomes is consid ered. The fourth part includes chapters related to glycan binding proteins. The first chapter covers history of dis covery and classification of glycan binding proteins. The following chapters discuss data about various types of lectins: R , L , P , C , and I type lectins. Here also galectins, microbial lectins, and proteins that bind sulfat ed glycosaminoglycans are considered. The fifth part is devoted to the role of glycans in physiology and disease. The role of glycans in glycopro tein quality control, their service as signaling molecules, and glycans in development and systematic physiology are discussed. Special chapters describe the role of gly cans in bacterial, viral, and parasitic infections. Also there is information about genetic disorders of glycan degrada tion (lysosomal storage diseases), glycosylation, and dis orders related to glycosylation changes in cancer. The final, sixth part of book highlights methods and applications used for study of glycans. Methods where antibodies and lectins are applied for study of glycans, chemical and enzymatic synthesis of glycans and glyco conjugates, chemical tools for inhibiting glycosylation, and glycans in biotechnology and pharmaceutical indus try are analyzed. Each chapter contains a short list of references. The book also includes a list of fundamental monographs dealing with various aspects of glycobiology. At the end of book there is study guide consisting of very provocative questions related to each chapter covering the major ideas expressed in the text. A dictionary of terms and a subject index are very helpful for readers. In general, the book is valuable not only for begin ners and experts in glycobiology, but also for many bio chemists, molecular biologists, biotechnologists, and physicians. ISSN 0006 2979, Biochemistry (Moscow), 2009, Vol. 74, No. 9, p. 1056. © Pleiades Publishing, Ltd., 2009. Published in Russian in Biokhimiya, 2009, published in Biokhimiya, 2009, Vol. 74, No. 9, p. 1294.
TL;DR: It is concluded that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.
Abstract: A commonly observed coupling of sensory domains to GGDEF-class diguanylate cyclases and EAL-class phosphodiesterases has long suggested that c-di-GMP synthesizing and degrading enzymes sense environmental signals. Nevertheless, relatively few signal ligands have been identified for these sensors, and even fewer instances of in vitro switching by ligand have been demonstrated. Here we describe an Escherichia coli two-gene operon, dosCP, for control of c-di-GMP by oxygen. In this operon, the gene encoding the oxygen-sensing c-di-GMP phosphodiesterase Ec Dos (here renamed Ec DosP) follows and is translationally coupled to a gene encoding a diguanylate cyclase, here designated DosC. We present the first characterizations of DosC and a detailed study of the ligand-dose response of DosP. Our results show that DosC is a globin-coupled sensor with an apolar but accessible heme pocket that binds oxygen with a K(d) of 20 microM. The response of DosP activation to increasing oxygen concentration is a complex function of its ligand saturation such that over 80% of the activation occurs in solutions that exceed 30% of air saturation (oxygen >75 microM). Finally, we find that DosP and DosC associate into a functional complex. We conclude that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.
TL;DR: Analytical, spectroscopic, and mutagenesis data indicate that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.
Abstract: The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S]2+ cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV−visible absorption and CD, resonance Raman, EPR, ENDOR, Mossbauer, and EXAFS studies of [2Fe-2S] Grx3/4...
TL;DR: Progress made in the field of degradomics is reviewed and a current view of the MMP degradome is presented, showing the metamorphosis of MMPs into modulators of multiple signaling pathways has been facilitated.
Abstract: Shotgun proteomics techniques are conceptually unbiased, but data interpretation and follow-up experiments are often constrained by dogma, established beliefs that are accepted without question, that can dilute the power of proteomics and hinder scientific progress. Proteomics and degradomics, the characterization of all proteases, inhibitors, and protease substrates by genomic and proteomic techniques, have exponentially expanded the known substrate repertoire of the matrix metalloproteinases (MMPs), even to include intracellular proteins with newly recognized extracellular functions. Thus, the dogma that MMPs are dowdy degraders of extracellular matrix has been resolutely overturned, and the metamorphosis of MMPs into modulators of multiple signaling pathways has been facilitated. Here we review progress made in the field of degradomics and present a current view of the MMP degradome.
TL;DR: Evidence that alpha-Synuclein forms a bent-helix when bound to highly curved detergent micelles, whereas it binds more physiological 100 nm diameter lipid vesicles as an elongated helix is found.
Abstract: Interactions between the synaptic protein α-Synuclein and cellular membranes may be relevant both to its native function as well as its role in Parkinson’s disease. We use single molecule Forster resonance energy transfer to probe the structure of α-Synuclein bound to detergent micelles and lipid vesicles. We find evidence that it forms a bent-helix when bound to highly curved detergent micelles, whereas it binds more physiological 100 nm diameter lipid vesicles as an elongated helix. Our results highlight the influence of membrane curvature in determining α-Synuclein conformation, which may be important for both its normal and disease-associated functions.
TL;DR: Niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated beta-catenin stabilization and LEF/TCF reporter activity, and may provide a valuable means of studying the physiological consequences of Wnt signaling.
Abstract: Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and stem cell related tissue-regenerative effects of Wnt signaling. Dysregulated Wnt signaling is associated with many cancers. Currently, there are no drug candidates or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated beta-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide-mediated internalization, the Frizzled1 receptor colocalizes in vesicles containing transferrin and agonist-activated beta(2)-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting upstream signaling molecules (i.e., Frizzled and Dishevelled) and moreover may provide a valuable means of studying the physiological consequences of Wnt signaling.
TL;DR: Testing for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D and extended X-ray absorption fine structure spectroscopy calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
Abstract: Aliphatic halogenases activate O2, cleave α-ketoglutarate (αKG) to CO2 and succinate, and form haloferryl [X−Fe(IV)═O; X = Cl or Br] complexes that cleave aliphatic C−H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related αKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O2 to form the C−H bond-cleaving ferryl complex is “triggered” by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presenc...
TL;DR: A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu( 2+) is central to Abeta neurotoxicity.
Abstract: Cu(2+) ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, l-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.
TL;DR: Recent advances in biochemical aspects of prion research are described, with a special focus on the mechanism of conversion ofPrP protein to the pathogenic form(s), the emerging structural knowledge of fungal and mammalian prions, and the rapidly growing understanding of the molecular basis of prions strains and their relation to barriers of interspecies transmissibility are described.
Abstract: Prion diseases, also known as the transmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative disorders that affect humans and animals. These diseases are intimately associated with conformational conversion of the cellular prion protein, PrP(C), into an oligomeric beta-sheet-rich form, PrP(Sc). A growing number of observations support the once heretical hypothesis that transmission of TSE diseases does not require nucleic acids and that PrP(Sc) alone can act as an infectious agent. The view that misfolded proteins can be infectious is also supported by recent findings regarding prion phenomena in yeast and other fungi. One of the most intriguing facets of prions is their ability to form different strains, leading to distinct phenotypes of TSE diseases. Within the context of the "protein-only" model, prion strains are believed to be encoded in distinct conformations of misfolded prion protein aggregates. In this review, we describe recent advances in biochemical aspects of prion research, with a special focus on the mechanism of conversion of prion protein to the pathogenic form(s), the emerging structural knowledge of fungal and mammalian prions, and our rapidly growing understanding of the molecular basis of prion strains and their relation to barriers of interspecies transmissibility.
TL;DR: Biophysical and cell biological approaches are beginning to elucidate the properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myelin.
Abstract: Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly wrapped multilamellar structure around neurons enabling rapid and efficient signal propagation. The gene of oligodendrocyte lineage (golli) gives rise to a variety of developmentally regulated splice isoforms of myelin basic protein (MBP), denoted golli for early forms and classic for later ones. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa; its central role is to maintain the structural integrity of the myelin sheath, by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane in a tight, spiral, multilamellar arrangement. This protein's extreme physicochemical properties, net charge of +19 at neutral pH, low proportion of hydrophobic residues, alternating regions of predicted intrinsic disorder and order, induced folding upon association with membranes and other proteins, and diversification via combinatorial post-translational modifications, define not only its role as a molecular Velcro in compact myelin, but as a multifunctional hub that may also bind to a number of other proteins and small molecule ligands in myelinating oligodendrocytes. In particular, MBP may link the underlying cytoskeleton and proteins containing SH3 domains to the membrane, allowing it to transduce transmembrane signals to the cytosol. These associations are facilitated by MBP being an intrinsically disordered protein, creating a large effective protein surface, and by the formation of transient and/or induced ordered secondary structure elements for molecular recognition. These processes can be modulated by a molecular barcode of numerous post-translational modifications and interactions with proteins such as calmodulin. In the human demyelinating disease multiple sclerosis, an aberrant pattern of modifications may contribute to demyelination and confound inherent attempts at repair. The conformational dynamics of the various isoforms and modified variants of MBP and their interactions with other proteins potentially allow them to participate in events coupling extracellular signals to cytoskeletal organization during myelination or remyelination. Various biophysical and cell biological approaches are beginning to elucidate these properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myelin.
TL;DR: Evidence is provided for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD.
Abstract: N-Terminally truncated and pyroglutamate (pGlu) modified amyloid beta (Abeta) peptides are major constituents of amyloid deposits in sporadic and inherited Alzheimer's disease (AD). Formation of pGlu at the N-terminus confers resistance against cleavage by most aminopeptidases, increases toxicity of the peptides, and may seed Abeta aggregate formation. Similarly, the deposited amyloid peptides ABri and ADan, which cause a very similar histopathology in familial British dementia (FBD) and familial Danish dementia (FDD), are N-terminally blocked by pGlu. Triggered by the coincidence of pGlu-modified amyloid peptides and similar pathology in AD, FBD, and FDD, we investigated the impact of N-terminal pGlu on biochemical and biophysical properties of Abeta, ABri, and ADan. N-Terminal pGlu increases the hydrophobicity and changes the pH-dependent solubility profile, rendering the pGlu-modified peptides less soluble in the basic pH range. The pGlu residue increases the aggregation propensity of all amyloid peptides as evidenced by ThT fluorescence assays and dynamic light scattering. The far-UV CD spectroscopic analysis points toward an enhanced beta-sheet structure of the pGlu-Abeta. Importantly, changes in fibril morphology are clearly caused by the N-terminal pGlu, resulting in the formation of short fibers, which are frequently arranged in bundles. The effect of pGlu on the morphology is virtually indistinguishable between ABri, ADan, and Abeta. The data provide evidence for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD. The main driving force for the aggregation is apparently an increase in the hydrophobicity and thus an accelerated seed formation.
TL;DR: With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers and has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.
Abstract: Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2 Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy
TL;DR: A method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome is developed, based on the affinity of this complex for the UBL-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif.
Abstract: Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions.
TL;DR: Spider venom, a factor that has played a decisive role in the evolution of one of the most successful groups of living organisms, is reviewed and special attention is given to the structure, properties, and biosynthesis of toxins of polypeptide nature.
Abstract: Spider venom, a factor that has played a decisive role in the evolution of one of the most successful groups of living organisms, is reviewed. Unique molecular diversity of venom components including substances of variable structure (from simple low molecular weight compounds to large multidomain proteins) with different functions is considered. Special attention is given to the structure, properties, and biosynthesis of toxins of polypeptide nature.
TL;DR: It is proposed that the reported ADP-ribosyltransferase activity of sirtuins is likely some inefficient side reactions of the deacetylase activity and may not be physiologically relevant.
Abstract: Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) onto specific target proteins. Sirtuins, a class of enzymes with NAD-dependent deacetylase activity, have been reported to possess ADP-ribosyltransferase activity, too. Here we used NAD analogues and 32P-NAD to study the ADP-ribosyltransferase activity of several different sirtuins, including yeast Sir2, human SirT1, mouse SirT4, and mouse SirT6. The results showed that an alkyne-tagged NAD is the substrate for deacetylation reactions but cannot detect the ADP-ribosylation activity. Furthermore, comparing with a bacterial ADP-ribosyltransferase diphtheria toxin, the observed rate constant of sirtuin-dependent ADP-ribosylation is >5000-fold lower. Compared with the kcat/Km values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is ∼500 times weaker. The weak ADP-ribosylation events can be explained by both e...
TL;DR: This review systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins.
Abstract: In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special "label" can get into it. A polyubiquitin chain plays the role of the "label": degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the "labeled" molecule, it is released and labels another molecule. This process has been named "Ub-dependent protein degradation". In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers' attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.
TL;DR: The complete kinase domain has been crystallized and its X-ray crystal structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.
Abstract: MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35−382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-γS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-γS with compound 1 and ADP w...
TL;DR: Solid-state NMR data indicate that guanidinium-phosphate interactions exist in penetratin, and guanIDinium groups play a stronger structural role than ammonium groups in the lipid-assisted translocation of CPPs across liquid-crystalline cell membranes.
Abstract: Cell-penetrating peptides (CPPs) are small cationic peptides that cross the cell membrane while carrying macromolecular cargoes. We use solid-state NMR to investigate the structure and lipid interaction of two cationic residues, Arg10 and Lys13, in the CPP penetratin. 13C chemical shifts indicate that Arg10 adopts a rigid β-strand conformation in the liquid-crystalline state of anionic lipid membranes. This behavior contrasts with all other residues observed so far in this peptide, which adopt a dynamic β-turn conformation with coil-like chemical shifts at physiological temperature. Low-temperature 13C−31P distances between the peptide and the lipid phosphates indicate that both the Arg10 guanidinium Cζ atom and the Lys13 Ce atom are close to the lipid 31P (4.0−4.2 A), proving the existence of charge−charge interaction for both Arg10 and Lys13 in the gel-phase membrane. However, since lysine substitution in CPPs is known to weaken their translocation ability, we propose that the low temperature stabilizes...
TL;DR: It is concluded that A22 is a competitive inhibitor of ATP binding to MreB, which represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.
Abstract: S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly propert...