Showing papers in "Annual Review of Biochemistry in 2014"

Intrinsically disordered proteins and intrinsically disordered protein regions.

Abstract: Intrinsically disordered proteins (IDPs) and IDP regions fail to form a stable structure, yet they exhibit biological activities. Their mobile flexibility and structural instability are encoded by their amino acid sequences. They recognize proteins, nucleic acids, and other types of partners; they accelerate interactions and chemical reactions between bound partners; and they help accommodate posttranslational modifications, alternative splicing, protein fusions, and insertions or deletions. Overall, IDP-associated biological activities complement those of structured proteins. Recently, there has been an explosion of studies on IDP regions and their functions, yet the discovery and investigation of these proteins have a long, mostly ignored history. Along with recent discoveries, we present several early examples and the mechanisms by which IDPs contribute to function, which we hope will encourage comprehensive discussion of IDPs and IDP regions in biochemistry textbooks. Finally, we propose future directions for IDP research.

The Scanning Mechanism of Eukaryotic Translation Initiation

Abstract: In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.

Demystifying Heparan Sulfate–Protein Interactions

Abstract: Numerous proteins, including cytokines and chemokines, enzymes and enzyme inhibitors, extracellular matrix proteins, and membrane receptors, bind heparin. Although they are traditionally classified as heparin-binding proteins, under normal physiological conditions these proteins actually interact with the heparan sulfate chains of one or more membrane or extracellular proteoglycans. Thus, they are more appropriately classified as heparan sulfate-binding proteins (HSBPs). This review provides an overview of the various modes of interaction between heparan sulfate and HSBPs, emphasizing biochemical and structural insights that improve our understanding of the many biological functions of heparan sulfate.

Biosynthesis and Export of Bacterial Lipopolysaccharides

Abstract: Lipopolysaccharide molecules represent a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in host-pathogen interplay. Few bacteria contain lipopolysaccharide molecules composed only of lipid A. In most forms, lipid A is glycosylated by addition of the core oligosaccharide that, in some bacteria, provides an attachment site for a long-chain O-antigenic polysaccharide. The complexity of lipopolysaccharide structures is reflected in the processes used for their biosynthesis and export. Rapid growth and cell division depend on the bacterial cell's capacity to synthesize and export lipopolysaccharide efficiently and in large amounts. We review recent advances in those processes, emphasizing the reactions that are essential for viability.

Genome Engineering with Targetable Nucleases

Abstract: Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for t...

Human RecQ helicases in DNA repair, recombination, and replication.

Abstract: RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability.

Expanding and Reprogramming the Genetic Code of Cells and Animals

Abstract: Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Understanding Nucleic Acid–Ion Interactions

Abstract: Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

Small Proteins Can No Longer Be Ignored

Abstract: Small proteins, here defined as proteins of 50 amino acids or fewer in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years, however, increasing numbers of small proteins have been identified either through the realization that mutations in intergenic regions are actually within unannotated small protein genes or through the discovery that some small, regulatory RNAs encode small proteins. These insights, together with comparative sequence analysis, indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review summarizes what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which these small proteins act and suggesting several general conclusions. Important questions for future studies of these overlooked proteins are also discussed.

Mechanism and Function of Oxidative Reversal of DNA and RNA Methylation

Abstract: The importance of eukaryotic DNA methylation [5-methylcytosine (5mC)] in transcriptional regulation and development was first suggested almost 40 years ago. However, the molecular mechanism underlying the dynamic nature of this epigenetic mark was not understood until recently, following the discovery that the TET proteins, a family of AlkB-like Fe(II)/α-ketoglutarate-dependent dioxygenases, can oxidize 5mC to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Since then, several mechanisms that are responsible for processing oxidized 5mC derivatives to achieve DNA demethylation have emerged. Our biochemical understanding of the DNA demethylation process has prompted new investigations into the biological functions of DNA demethylation. Characterization of two additional AlkB family proteins, FTO and ALKBH5, showed that they possess demethylase activity toward N6-methyladenosine (m6A) in RNA, indicating that members of this subfamily of dioxygenases have a gene...

Activity-based profiling of proteases.

Abstract: Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

Enzyme Inhibitor Discovery by Activity-Based Protein Profiling

Abstract: Eukaryotic and prokaryotic organisms possess huge numbers of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biological systems. Here, we discuss how the chemical proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo–active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochemical and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiology and disease. These studies demonstrate the value of selective chemical probes as drivers of biological inquiry.

Histone chaperones: assisting histone traffic and nucleosome dynamics.

Abstract: The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

Progress Toward Synthetic Cells

Abstract: The complexity of even the simplest known life forms makes efforts to synthesize living cells from inanimate components seem like a daunting task. However, recent progress toward the creation of synthetic cells, ranging from simple protocells to artificial cells approaching the complexity of bacteria, suggests that the synthesis of life is now a realistic goal. Protocell research, fueled by advances in the biophysics of primitive membranes and the chemistry of nucleic acid replication, is providing new insights into the origin of cellular life. Parallel efforts to construct more complex artificial cells, incorporating translational machinery and protein enzymes, are providing information about the requirements for protein-based life. We discuss recent advances and remaining challenges in the synthesis of artificial cells, the possibility of creating new forms of life distinct from existing biology, and the promise of this research for gaining a deeper understanding of the nature of living systems.

Lipidomics: analysis of the lipid composition of cells and subcellular organelles by electrospray ionization mass spectrometry.

Abstract: Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems. Lipidomics has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Here, I review state-of-the-art electrospray ionization tandem mass spectrometric approaches and direct infusion to quantitatively assess lipid compositions of cells and subcellular fractions. Finally, I discuss a few examples of the power of mass spectrometry–based lipidomics in addressing cell biological questions.

RNA helicase proteins as chaperones and remodelers.

Abstract: Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are the promotion of rearrangements of structured RNAs and the remodeling of ribonucleoprotein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. Although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA, and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of thei...

Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR

Abstract: Large macromolecular assemblies, so-called molecular machines, are critical to ensuring proper cellular function. Understanding how proper function is achieved at the atomic level is crucial to advancing multiple avenues of biomedical research. Biophysical studies often include X-ray diffraction and cryo-electron microscopy, providing detailed structural descriptions of these machines. However, their inherent flexibility has complicated an understanding of the relation between structure and function. Solution NMR spectroscopy is well suited to the study of such dynamic complexes, and continued developments have increased size boundaries; insights into function have been obtained for complexes with masses as large as 1 MDa. We highlight methyl-TROSY (transverse relaxation optimized spectroscopy) NMR, which enables the study of such large systems, and include examples of applications to several cellular machines. We show how this emerging technique contributes to an understanding of cellular function and the role of molecular plasticity in regulating an array of biochemical activities.

Regulating the chromatin landscape: structural and mechanistic perspectives.

Abstract: A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).

Chlorophyll Modifications and Their Spectral Extension in Oxygenic Photosynthesis

Abstract: Chlorophylls are magnesium-tetrapyrrole molecules that play essential roles in photosynthesis. All chlorophylls have similar five-membered ring structures, with variations in the side chains and/or reduction states. Formyl group substitutions on the side chains of chlorophyll a result in the different absorption properties of chlorophyll b, chlorophyll d, and chlorophyll f. These formyl substitution derivatives exhibit different spectral shifts according to the formyl substitution position. Not only does the presence of various types of chlorophylls allow the photosynthetic organism to harvest sunlight at different wavelengths to enhance light energy input, but the pigment composition of oxygenic photosynthetic organisms also reflects the spectral properties on the surface of the Earth. Two major environmental influencing factors are light and oxygen levels, which may play central roles in the regulatory pathways leading to the different chlorophylls. I review the biochemical processes of chlorophyll biosynthesis and their regulatory mechanisms.

Selection-based discovery of druglike macrocyclic peptides.

Abstract: Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.

Hierarchy of RNA functional dynamics

Abstract: RNA dynamics play a fundamental role in many cellular functions. However, there is no general framework to describe these complex processes, which typically consist of many structural maneuvers that occur over timescales ranging from picoseconds to seconds. Here, we classify RNA dynamics into distinct modes representing transitions between basins on a hierarchical free-energy landscape. These transitions include large-scale secondary-structural transitions at >0.1-s timescales, base-pair/tertiary dynamics at microsecond-to-millisecond timescales, stacking dynamics at timescales ranging from nanoseconds to microseconds, and other “jittering” motions at timescales ranging from picoseconds to nanoseconds. We review various modes within these three different tiers, the different mechanisms by which they are used to regulate function, and how they can be coupled together to achieve greater functional complexity.

High-Resolution Structure of the Eukaryotic 80S Ribosome

Abstract: The high-resolution structure of the eukaryotic ribosome from yeast, determined at 3.0-A resolution, permitted the unambiguous determination of the protein side chains, eukaryote-specific proteins, protein insertions, and ribosomal RNA expansion segments of the 80 proteins and ∼5,500 RNA bases that constitute the 80S ribosome. A comparison between this first atomic model of the entire 80S eukaryotic ribosome and previously determined structures of bacterial ribosomes confirmed early genetic and structural data indicating that they share an evolutionarily conserved core of ribosomal RNA and proteins. It also confirmed the conserved organization of essential functional sites, such as the peptidyl transferase center and the decoding site. New structural information about eukaryote-specific elements, such as expansion segments and new ribosomal proteins, forms the structural framework for the design and analysis of experiments that will explore the eukaryotic translational apparatus and the evolutionary force...

Interactive Features of Proteins Composing Eukaryotic Circadian Clocks

Abstract: Research into the molecular mechanisms of eukaryotic circadian clocks has proceeded at an electrifying pace. In this review, we discuss advances in our understanding of the structures of central molecular players in the timing oscillators of fungi, insects, and mammals. A series of clock protein structures demonstrate that the PAS (Per/Arnt/Sim) domain has been used with great variation to formulate the transcriptional activators and repressors of the clock. We discuss how posttranslational modifications and external cues, such as light, affect the conformation and function of core clock components. Recent breakthroughs have also revealed novel interactions among clock proteins and new partners that couple the clock to metabolic and developmental pathways. Overall, a picture of clock function has emerged wherein conserved motifs and structural platforms have been elaborated into a highly dynamic collection of interacting molecules that undergo orchestrated changes in chemical structure, conformational state, and partners.

Metabolic and Nontranscriptional Circadian Clocks: Eukaryotes

Abstract: Circadian clocks are cellular timekeeping mechanisms that coordinate behavior and physiology around the 24-h day in most living organisms. Misalignment of an organism's clock with its environment is associated with long-term adverse fitness consequences, as exemplified by the link between circadian disruption and various age-related diseases in humans. Current eukaryotic models of the circadian oscillator rely on transcription/translation feedback loop mechanisms, supplemented with accessory cytosolic loops that connect them to cellular physiology. However, mounting evidence is questioning the absolute necessity of transcription-based oscillators for circadian rhythmicity, supported by the recent discovery of oxidation-reduction cycles of peroxiredoxin proteins, which persist even in the absence of transcription. A more fundamental mechanism based on metabolic cycles could thus underlie circadian transcriptional and cytosolic rhythms, thereby promoting circadian oscillations to integral properties of cell...

Topological regulation of lipid balance in cells.

Abstract: Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient.

Metabolic Compensation and Circadian Resilience in Prokaryotic Cyanobacteria

Abstract: For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.

Asymmetry of Single Cells and Where That Leads

Abstract: Most single animal cells have an internal vector that determines where recycling membrane is added to the cell's surface. Because of the specific molecular composition of this added membrane, a dynamic asymmetry is formed on the surface of the cell. The consequences of this dynamic asymmetry are discussed, together with what they imply for how cells move. The polarity of a single-celled embryo, such as that of the nematode Caenorhabditis elegans, is explored in a similar framework.

Lipids and Extracellular Materials

Abstract: This article introduces the Lipids and Extracellular Materials theme of the Annual Review of Biochemistry, Volume 83.

Journeys in science: glycobiology and other paths.

Abstract: My scientific journeys began at Oxford nearly 50 years ago. My paths have taken me from magnetic resonance through enzyme systems to antibodies, which led directly to glycobiology. Oxford University's first industrial grant helped the development of the technology for isolating and sequencing oligosaccharides from glycoproteins. This technology was disseminated through a spin-off company, Oxford GlycoSystems, and by the establishment of the Glycobiology Institute. The technology gave rise to the concept of glycoforms, which allow diversification of a protein's properties. Iminosugars, which are glucosidase inhibitors, can interfere with the initial steps of glycan processing on proteins and inhibit three-dimensional folding of glycoproteins. Glucosidase targets for therapy include viral envelope glycoproteins. Clinical trials of an iminosugar as an antiviral for dengue virus are under way. Another iminosugar activity, inhibition of glycolipid synthesis, resulted in a drug for Gaucher disease, which was ap...

Dynamics and Timekeeping in Biological Systems

Abstract: This article introduces three reviews on the theme of circadian rhythms.