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Annual Review of Biochemistry - Volume 80, 2011
Volume 80, 2011
- Preface
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From Serendipity to Therapy
Vol. 80 (2011), pp. 1–15More LessMy postdoctoral training in the biosynthesis of plant polysaccharides at the University of California, Berkeley, led me, rather improbably, to study mucopolysaccharide storage disorders in the intramural program of the National Institutes of Health (NIH). I have traced the path from studies of mucopolysaccharide turnover in cultured cells to the development of therapy for patients. The key experiment started as an accident, i.e., the mixing of cells of different genotypes, resulting in correction of their biochemical defect. This serendipitous experiment led to identification of the enzyme deficiencies in the Hurler and Hunter syndromes, to an understanding of the biochemistry of lysosomal enzymes in general, and to the cell biology of receptor-mediated endocytosis and targeting to lysosomes. It paved the way for the development of enzyme replacement therapy with recombinant enzymes. I have also included studies performed after I moved to the University of California, Los Angeles (UCLA), including a recent unexpected finding in a neurodegenerative mucopolysaccharide storage disease, the Sanfilippo syndrome, with implications for therapy.
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Journey of a Molecular Biologist
Vol. 80 (2011), pp. 16–40More LessMy journey into a research career began in fermentation biochemistry in an applied science department during the difficult post-World War II time in Japan. Subsequently, my desire to do research in basic science developed. I was fortunate to be a postdoctoral fellow in the United States during the early days of molecular biology. From 1957 to 1960, I worked with three pioneers of molecular biology, Sol Spiegelman, James Watson, and Seymour Benzer. These experiences helped me develop into a basic research scientist. My initial research projects at Osaka University, and subsequently at the University of Wisconsin, Madison, were on the mode of action of colicins as well as on mRNA and ribosomes. Following success in the reconstitution of ribosomal subunits, my efforts focused more on ribosomes, initially on the aspects of structure, function, and in vitro assembly, such as the construction of the 30S subunit assembly map. After this, my laboratory studied the regulation of the synthesis of ribosomes and ribosomal components in Escherichia coli. Our achievements included the discovery of translational feedback regulation of ribosomal protein synthesis and the identification of several repressor ribosomal proteins used in this regulation. In 1984, I moved to the University of California, Irvine, and initiated research on rRNA transcription by RNA polymerase I in the yeast Saccharomyces cerevisiae. The use of yeast genetics combined with biochemistry allowed us to identify genes uniquely involved in rRNA synthesis and to elucidate the mechanism of initiation of transcription. This essay is a reflection on my life as a research scientist.
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My Life with Nature
Vol. 80 (2011), pp. 42–70More LessAfter a childhood in Germany and being a youth in Grand Forks, North Dakota, I went to Harvard University, then to graduate school in biochemistry at the University of Wisconsin. Then to Washington University and Stanford University for postdoctoral training in biochemistry and genetics. Then at the University of Wisconsin, as a professor in the Department of Biochemistry and the Department of Genetics, I initiated research on bacterial chemotaxis. Here, I review this research by me and by many, many others up to the present moment. During the past few years, I have been studying chemotaxis and related behavior in animals, namely in Drosophila fruit flies, and some of these results are presented here. My current thinking is described.
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Protein Folding and Modification in the Mammalian Endoplasmic Reticulum
Vol. 80 (2011), pp. 71–99More LessAnalysis of the human genome reveals that approximately a third of all open reading frames code for proteins that enter the endoplasmic reticulum (ER), demonstrating the importance of this organelle for global protein maturation. The path taken by a polypeptide through the secretory pathway starts with its translocation across or into the ER membrane. It then must fold and be modified correctly in the ER before being transported via the Golgi apparatus to the cell surface or another destination. Being physically segregated from the cytosol means that the ER lumen has a distinct folding environment. It contains much of the machinery for fulfilling the task of protein production, including complex pathways for folding, assembly, modification, quality control, and recycling. Importantly, the compartmentalization means that several modifications that do not occur in the cytosol, such as glycosylation and extensive disulfide bond formation, can occur to secreted proteins to enhance their stability before their exposure to the extracellular milieu. How these various machineries interact during the normal pathway of folding and protein secretion is the subject of this review.
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Mechanisms of Membrane Curvature Sensing
Vol. 80 (2011), pp. 101–123More LessBacteria and eukaryotic cells contain geometry-sensing tools in their cytosol: protein motifs or domains that recognize the curvature, concave or convex, deep or shallow, of lipid membranes. These sensors contrast with classical lipid-binding domains by their extended structure and, sometimes, counterintuitive chemistry. Among the sensors are long amphipathic helices, such as the ALPS motif and the N-terminal region of α-synuclein, whose apparent “design defects” translate into a remarkable ability to specifically adsorb to the surface of small vesicles. Fundamental differences in the lipid composition of membranes of the early and late secretory pathways probably explain why some sensors use mostly electrostatics whereas others take advantage of the hydrophobic effect. Membrane curvature sensors help to organize very diverse reactions, such as lipid transfer between membranes, the tethering of vesicles at the Golgi apparatus, and the assembly-disassembly cycle of protein coats.
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Biogenesis and Cargo Selectivity of Autophagosomes
Vol. 80 (2011), pp. 125–156More LessAutophagy is a major catabolic pathway in eukaryotes, which is required for the lysosomal/vacuolar degradation of cytoplasmic proteins and organelles. Interest in the autophagy pathway has recently gained momentum largely owing to identification of multiple autophagy-related genes and recognition of its involvement in various physiological conditions. Here we review current knowledge of the molecular mechanisms regulating autophagy in mammals and yeast, specifically the biogenesis of autophagosomes and the selectivity of their cargo recruitment. We discuss the different steps of autophagy, from the signal transduction events that regulate it to the completion of this pathway by fusion with the lysosome/vacuole. We also review research on the origin of the autophagic membrane, the molecular mechanism of autophagosome formation, and the roles of two ubiquitin-like protein families and other structural elements that are essential for this process. Finally, we discuss the various modes of autophagy and highlight their functional relevance for selective degradation of specific cargos.
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Introduction to Theme “Membrane Protein Folding and Insertion”
Vol. 80 (2011), pp. 157–160More LessThis volume of the Annual Review of Biochemistry contains three reviews on current developments in membrane protein research: Grigoryan et al. “Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K+ Channels, and Integrin Receptors,” Hagan et al. “β-Barrel Membrane Protein Assembly by the Bam Complex,” and Dalbey et al. “Assembly of Bacterial Inner Membrane Proteins.” In this short introduction, I discuss these reviews in the larger context of where the field of membrane protein biochemistry is heading.
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Assembly of Bacterial Inner Membrane Proteins
Vol. 80 (2011), pp. 161–187More LessNumerous membrane proteins form multisubunit protein complexes, which contain both integral and peripheral subunits, in addition to prosthetic groups. Bacterial membrane proteins are inserted into the inner membrane by the Sec translocase and YidC insertase. Their folding can be facilitated by YidC and the phospholipid phosphatidylethanolamine (PE). Glycine zippers and other motifs promote transmembrane-transmembrane (TM-TM) helix interactions that may lead to the formation of α-helical bundles of membrane proteins. During or after membrane insertion, the subunits of oligomeric membrane proteins must find each other to build the homo-oligomeric and the hetero-oligomeric membrane complexes. Although chaperones may function as assembly factors in the formation of the oligomer, many protein oligomers appear to fold and oligomerize spontaneously. Current studies show that most subunits of hetero-oligomers follow a sequential and ordered pathway to form the membrane protein complex. If the inserted protein is misfolded or the membrane protein is misassembled, quality control mechanisms exist that can degrade the proteins.
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β-Barrel Membrane Protein Assembly by the Bam Complex
Vol. 80 (2011), pp. 189–210More Lessβ-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.
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Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K+ Channels, and Integrin Receptors
Vol. 80 (2011), pp. 211–237More LessSignal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems.
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Mass Spectrometry in the Postgenomic Era
Vol. 80 (2011), pp. 239–246More LessMass spectrometry (MS) is rapidly becoming an essential tool for biologists and biochemists in their efforts to throw light on molecular mechanisms within cellular systems. Used in unison with genome sequence data, MS has developed into the method of choice for identifying proteins, elucidating their posttranslational modifications, and reading out their functional interactions. Variations of the method have even begun to enable accurate mass determination of intact protein complexes, allowing for direct determination of subunit stoichiometry and the interactions between the subunits. Advances in mass spectrometric technologies have also led to great improvements in our ability to probe and define many of the other key molecular players in cells, including the all important lipid components. We provide here some perspectives on the explosion of applications of MS to protein science, systems biology, proteomics, lipidomics, and cell biology in general.
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Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes
Vol. 80 (2011), pp. 247–271More LessRapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.
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Quantitative, High-Resolution Proteomics for Data-Driven Systems Biology
Jürgen Cox, and Matthias MannVol. 80 (2011), pp. 273–299More LessSystems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem mass spectrometry (MS/MS) to identify and quantify peptides at a large scale. This peptide intensity information is the basic quantitative proteomic data type. It is used to quantify proteins between different proteome states, including the temporal variation of the proteome, to determine the complete primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far.
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Applications of Mass Spectrometry to Lipids and Membranes
Vol. 80 (2011), pp. 301–325More LessLipidomics, a major part of metabolomics, constitutes the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. As with proteomics, mass spectrometry has earned a central analytical role in lipidomics, and this role will continue to grow with technological developments. Currently, there exist two mass spectrometry–based lipidomics approaches, one based on a division of lipids into categories and classes prior to analysis, the “comprehensive lipidomics analysis by separation simplification” (CLASS), and the other in which all lipid species are analyzed together without prior separation, shotgun. In exploring the lipidome of various living systems, novel lipids are being discovered, and mass spectrometry is helping characterize their chemical structure. Deuterium exchange mass spectrometry (DXMS) is being used to investigate the association of lipids and membranes with proteins and enzymes, and imaging mass spectrometry (IMS) is being applied to the in situ analysis of lipids in tissues.
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Emerging In Vivo Analyses of Cell Function Using Fluorescence Imaging*
Vol. 80 (2011), pp. 327–332More LessUnderstanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.
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Biochemistry of Mobile Zinc and Nitric Oxide Revealed by Fluorescent Sensors
Vol. 80 (2011), pp. 333–355More LessBiological mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes.
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Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer
Vol. 80 (2011), pp. 357–373More LessMany genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein–based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.
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Reporting from the Field: Genetically Encoded Fluorescent Reporters Uncover Signaling Dynamics in Living Biological Systems
Sohum Mehta, and Jin ZhangVol. 80 (2011), pp. 375–401More LessReal-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena.
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DNA Replicases from a Bacterial Perspective
Vol. 80 (2011), pp. 403–436More LessBacterial replicases are complex, tripartite replicative machines. They contain a polymerase, polymerase III (Pol III), a β2 processivity factor, and a DnaX complex ATPase that loads β2 onto DNA and chaperones Pol III onto the newly loaded β2. Bacterial replicases are highly processive, yet cycle rapidly during Okazaki fragment synthesis in a regulated way. Many bacteria encode both a full-length τ and a shorter γ form of DnaX by a variety of mechanisms. γ appears to be uniquely placed in a single position relative to two τ protomers in a pentameric ring. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to a prototypical ε Mg2+-dependent exonuclease, but also contains a second Zn2+-dependent proofreading exonuclease, at least in some bacteria. This review focuses on a critical evaluation of recent literature and concepts pertaining to the above issues and suggests specific areas that require further investigation.
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Genomic and Biochemical Insights into the Specificity of ETS Transcription Factors
Vol. 80 (2011), pp. 437–471More LessETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5′-GGA(A/T)-3′. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.
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Signals and Combinatorial Functions of Histone Modifications
Vol. 80 (2011), pp. 473–499More LessAlterations of chromatin structure have been shown to be crucial for response to cell signaling and for programmed gene expression in development. Posttranslational histone modifications influence changes in chromatin structure both directly and by targeting or activating chromatin-remodeling complexes. Histone modifications intersect with cell signaling pathways to control gene expression and can act combinatorially to enforce or reverse epigenetic marks in chromatin. Through their recognition by protein complexes with enzymatic activities cross talk is established between different modifications and with other epigenetic pathways, including noncoding RNAs (ncRNAs) and DNA methylation. Here, we review the functions of histone modifications and their exploitation in the programming of gene expression during several events in development.
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Assembly of Bacterial Ribosomes
Vol. 80 (2011), pp. 501–526More LessThe assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.
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The Mechanism of Peptidyl Transfer Catalysis by the Ribosome
Vol. 80 (2011), pp. 527–555More LessThe ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the α-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions.
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Amyloid Structure: Conformational Diversity and Consequences
Vol. 80 (2011), pp. 557–585More LessMany, perhaps most, proteins, are capable of forming self-propagating, β-sheet (amyloid) aggregates. Amyloid-like aggregates are found in a wide range of diseases and underlie prion-based inheritance. Despite intense interest in amyloids, structural details have only recently begun to be revealed as advances in biophysical approaches, such as hydrogen-deuterium exchange, X-ray crystallography, solid-state nuclear magnetic resonance (SSNMR), and cryoelectron microscopy (cryoEM), have enabled high-resolution insights into their molecular organization. Initial studies found that despite the highly divergent primary structure of different amyloid-forming proteins, amyloids from different sources share many structural similarities. With higher-resolution information, however, it has become clear that, on the molecular level, amyloids comprise a wide diversity of structures. Particularly surprising has been the finding that identical polypeptides can fold into multiple, distinct amyloid conformations and that this structural diversity can lead to distinct heritable prion states or strains.
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AAA+ Proteases: ATP-Fueled Machines of Protein Destruction
Vol. 80 (2011), pp. 587–612More LessAAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates.
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The Structure of the Nuclear Pore Complex
Vol. 80 (2011), pp. 613–643More LessIn eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.
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Benchmark Reaction Rates, the Stability of Biological Molecules in Water, and the Evolution of Catalytic Power in Enzymes
Vol. 80 (2011), pp. 645–667More LessThe rates of enzyme reactions fall within a relatively narrow range. To estimate the rate enhancements produced by enzymes, and their expected affinities for transition state analog inhibitors, it is necessary to measure the rates of the corresponding reactions in water in the absence of a catalyst. This review describes the spontaneous cleavages of C-C, C-H, C-N, C-O, P-O, and S-O bonds in biological molecules, as well as the uncatalyzed reactions that correspond to phosphoryl transfer reactions catalyzed by kinases and to peptidyl transfer in the ribosome. The rates of these reactions, some with half-lives in excess of one million years, span an overall range of 1019-fold. Moreover, the slowest reactions tend to be most sensitive to temperature, with rates that increase as much as 107-fold when the temperature is raised from 25° to 100°C. That tendency collapses, by many orders of magnitude, the time that would have been required for chemical evolution on a warm earth. If the catalytic effect of primitive enzymes, like that of modern enzymes and many nonenzymatic catalysts, were mainly to reduce a reaction's enthalpy of activation, then the resulting rate enhancement would have increased automatically as the surroundings cooled. By reducing the time required for early chemical evolution in a warm environment, these findings counter the view that not enough time has passed for terrestrial life to have evolved to its present level of complexity.
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Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis
Vol. 80 (2011), pp. 669–702More LessPhosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.
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Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes
Vol. 80 (2011), pp. 703–732More LessExperimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein's dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.
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Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo
Vol. 80 (2011), pp. 733–767More LessIncorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an FeIIIFeIII-tyrosyl radical (Y•) cofactor (class Ia), a MnIIIMnIII-Y• cofactor (class Ib), and a MnIVFeIII cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.
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The Evolution of Protein Kinase Inhibitors from Antagonists to Agonists of Cellular Signaling
Vol. 80 (2011), pp. 769–795More LessKinases are highly regulated enzymes with diverse mechanisms controlling their catalytic output. Over time, chemical discovery efforts for kinases have produced ATP-competitive compounds, allosteric regulators, irreversible binders, and highly specific inhibitors. These distinct classes of small molecules have revealed many novel aspects about kinase-mediated signaling, and some have progressed from simple tool compounds into clinically validated therapeutics. This review explores several small-molecule inhibitors for kinases highlighting elaborate mechanisms by which kinase function is modulated. A complete surprise of targeted kinase drug discovery has been the finding of ATP-competitive inhibitors that behave as agonists, rather than antagonists, of their direct kinase target. These studies hint at a connection between ATP-binding site occupancy and networks of communication that are independent of kinase catalysis. Indeed, kinase inhibitors that induce changes in protein localization, protein-protein interactions, and even enhancement of catalytic activity of the target kinase have been found. The relevance of these findings to the therapeutic efficacy of kinase inhibitors and to the future identification of new classes of drug targets is discussed.
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Glycan Microarrays for Decoding the Glycome
Vol. 80 (2011), pp. 797–823More LessIn the last decade, glycan microarrays have revolutionized the analysis of the specificity of glycan-binding proteins (GBPs), providing information that simultaneously illuminates the biology mediated by them and decodes the informational content of the glycome. Numerous methods have emerged for arraying glycans in a “chip” format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of GBPs, which mediate mammalian biology, host-pathogen interactions, and immune recognition of glycans relevant to vaccine production and cancer antigens. This review covers the development of glycan microarrays and applications that have provided insights into the roles of mammalian and microbial GBPs.
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Cross Talk Between O-GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription, and Chronic Disease
Vol. 80 (2011), pp. 825–858More LessO-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.
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Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae
Vol. 80 (2011), pp. 859–883More LessThe yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products.
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Sterol Regulation of Metabolism, Homeostasis, and Development
Joshua Wollam, and Adam AntebiVol. 80 (2011), pp. 885–916More LessSterol metabolites are critical signaling molecules that regulate metabolism, development, and homeostasis. Oxysterols, bile acids (BAs), and steroids work primarily through cognate sterol-responsive nuclear hormone receptors to control these processes through feed-forward and feedback mechanisms. These signaling pathways are conserved from simple invertebrates to mammals. Indeed, results from various model organisms have yielded fundamental insights into cholesterol and BA homeostasis, lipid and glucose metabolism, protective mechanisms, tissue differentiation, development, reproduction, and even aging. Here, we review how sterols act through evolutionarily ancient mechanisms to control these processes.
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Structural Biology of the Toll-Like Receptor Family
Jin Young Kang, and Jie-Oh LeeVol. 80 (2011), pp. 917–941More LessInnate immune receptors respond to common structural patterns in microbial molecules and are called pattern recognition receptors. Toll-like receptors (TLRs) play critical roles in the innate immune system by recognizing microbial lipids, carbohydrates, nucleic acids, and proteins. Precise definition of the ligand “pattern” of TLRs has been difficult to determine primarily owing to a lack of high-resolution structures. Recently, the structures of several TLR-ligand complexes and the intracellular signaling domains have been determined by X-ray crystallography. This new structural information, combined with extensive biochemical and immunological data accumulated over decades, sheds new light on ligand-recognition and -activation mechanisms. In this review, we summarize the TLR structures and discuss proposed ligand-recognition and -activation mechanisms.
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Structure-Function Relationships of the G Domain, a Canonical Switch Motif
Vol. 80 (2011), pp. 943–971More LessGTP-binding (G) proteins constitute a class of P-loop (phosphate-binding loop) proteins that work as molecular switches between the GDP-bound OFF and the GTP-bound ON state. The common principle is the 160–180-residue G domain with an α,β topology that is responsible for nucleotide-dependent conformational changes and drives many biological functions. Although the G domain uses a universally conserved switching mechanism, its structure, function, and GTPase reaction are modified for many different pathways and processes.
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STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions
Vol. 80 (2011), pp. 973–1000More LessEukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.
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Amino Acid Signaling in TOR Activation
Vol. 80 (2011), pp. 1001–1032More LessThe target of rapamycin (TOR) is a central cell growth regulator conserved from yeast to mammals. Uncontrolled TOR activation is commonly observed in human cancers. TOR forms two distinct structural and functional complexes, TORC1 and TORC2. TORC1 promotes cell growth and cell size by stimulating protein synthesis. A wide range of signals, including nutrients, energy levels, and growth factors, are known to control TORC1 activity. Among them, amino acids (AA) not only potently activate TORC1 but are also required for TORC1 activation by other stimuli, such as growth factors. The mechanisms of growth factors and cellular energy status in activating TORC1 have been well elucidated, whereas the molecular basis of AA signaling is just emerging. Recent advances in the role of AA signaling on TORC1 activation have revealed key components, including the Rag GTPases, protein kinases, nutrient transporters, and the intracellular trafficking machinery, in relaying AA signals to TORC1 activation.
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Mitochondrial tRNA Import and Its Consequences for Mitochondrial Translation
Vol. 80 (2011), pp. 1033–1053More LessThe mitochondrial genomes of most eukaryotes lack a variable number of tRNA genes. This lack is compensated for by import of a small fraction of the corresponding cytosolic tRNAs. There are two broad mechanisms for the import of tRNAs into mitochondria. In the first one, the tRNA is coimported together with a mitochondrial precursor protein along the protein import pathway. It applies to the yeast tRNALys and has been elucidated in great detail. In the second more vaguely defined mechanism, which is mainly found in plants and protozoa, tRNAs are directly imported independent of cytosolic factors. However, results in plants indicate that direct import of tRNAs may nevertheless require some components of the protein import machinery. All imported tRNAs in all systems are of the eukaryotic type but need to be functionally integrated into the mitochondrial translation system of bacterial descent. For some tRNAs, this is not trivial and requires unique evolutionary adaptations.
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Caspase Substrates and Cellular Remodeling
Vol. 80 (2011), pp. 1055–1087More LessThe caspases are unique proteases that mediate the major morphological changes of apoptosis and various other cellular remodeling processes. As we catalog and study the myriad proteins subject to cleavage by caspases, we are beginning to appreciate the full functional repertoire of these enzymes. Here, we examine current knowledge about caspase cleavages: what kinds of proteins are cut, in what contexts, and to what end. After reviewing basic caspase biology, we describe the technologies that enable high-throughput caspase substrate discovery and the datasets they have yielded. We discuss how caspases recognize their substrates and how cleavages are conserved among different metazoan organisms. Rather than comprehensively reviewing all known substrates, we use examples to highlight some functional impacts of caspase cuts during apoptosis and differentiation. Finally, we discuss the roles caspase substrates can play in medicine. Though great progress has been made in this field, many important areas still await exploration.
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Regulation of HSF1 Function in the Heat Stress Response: Implications in Aging and Disease
Vol. 80 (2011), pp. 1089–1115More LessTo dampen proteotoxic stresses and maintain protein homeostasis, organisms possess a stress-responsive molecular machinery that detects and neutralizes protein damage. A prominent feature of stressed cells is the increased synthesis of heat shock proteins (Hsps) that aid in the refolding of misfolded peptides and restrain protein aggregation. Transcriptional activation of the heat shock response is orchestrated by heat shock factor 1 (HSF1), which rapidly translocates to hsp genes and induces their expression. Although the role of HSF1 in protecting cells and organisms against severe stress insults is well established, many aspects of how HSF1 senses qualitatively and quantitatively different forms of stresses have remained poorly understood. Moreover, recent discoveries that HSF1 controls life span have prompted new ways of thinking about an old transcription factor. Here, we review the established role of HSF1 in counteracting cell stress and prospect the role of HSF1 as a regulator of disease states and aging.
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Previous Volumes
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Volume 92 (2023)
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Volume 91 (2022)
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Volume 90 (2021)
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Volume 89 (2020)
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Volume 88 (2019)
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Volume 87 (2018)
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Volume 86 (2017)
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Volume 85 (2016)
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Volume 84 (2015)
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Volume 83 (2014)
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Volume 82 (2013)
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Volume 81 (2012)
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Volume 80 (2011)
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Volume 79 (2010)
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Volume 78 (2009)
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Volume 77 (2008)
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Volume 76 (2007)
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Volume 75 (2006)
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Volume 74 (2005)
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Volume 73 (2004)
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Volume 72 (2003)
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Volume 71 (2002)
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Volume 70 (2001)
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Volume 69 (2000)
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Volume 68 (1999)
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Volume 67 (1998)
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Volume 66 (1997)
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Volume 65 (1996)
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Volume 64 (1995)
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Volume 63 (1994)
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Volume 62 (1993)
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Volume 61 (1992)
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Volume 60 (1991)
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Volume 59 (1990)
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Volume 58 (1989)
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Volume 57 (1988)
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Volume 56 (1987)
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Volume 55 (1986)
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Volume 54 (1985)
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Volume 53 (1984)
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Volume 52 (1983)
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Volume 51 (1982)
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Volume 50 (1981)
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Volume 49 (1980)
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Volume 48 (1979)
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Volume 47 (1978)
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Volume 46 (1977)
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Volume 45 (1976)
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Volume 44 (1975)
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Volume 43 (1974)
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Volume 42 (1973)
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Volume 41 (1972)
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Volume 40 (1971)
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Volume 39 (1970)
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Volume 38 (1969)
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Volume 37 (1968)
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Volume 36 (1967)
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Volume 35 (1966)
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Volume 34 (1965)
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Volume 33 (1964)
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Volume 32 (1963)
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Volume 31 (1962)
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Volume 30 (1961)
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Volume 29 (1960)
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Volume 28 (1959)
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Volume 27 (1958)
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Volume 26 (1957)
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Volume 25 (1956)
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Volume 24 (1955)
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Volume 23 (1954)
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Volume 22 (1953)
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Volume 21 (1952)
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Volume 20 (1951)
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Volume 19 (1950)
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Volume 18 (1949)
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Volume 17 (1948)
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Volume 16 (1947)
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Volume 15 (1946)
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Volume 14 (1945)
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Volume 13 (1944)
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Volume 12 (1943)
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Volume 11 (1942)
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Volume 10 (1941)
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Volume 9 (1940)
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Volume 8 (1939)
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Volume 7 (1938)
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Volume 6 (1937)
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Volume 5 (1936)
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Volume 4 (1935)
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Volume 3 (1934)
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Volume 2 (1933)
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Volume 1 (1932)
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Volume 0 (1932)