Showing papers in "Annual Review of Biochemistry in 2008"
TL;DR: Recent advances in the understanding of the diverse biological actions of PPARgamma are reviewed with an eye toward the expanding therapeutic potential of PPargamma agonist drugs.
Abstract: The nuclear receptor PPARgamma is a ligand-activated transcription factor that plays an important role in the control of gene expression linked to a variety of physiological processes. PPARgamma was initially characterized as the master regulator for the development of adipose cells. Ligands for PPARgamma include naturally occurring fatty acids and the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma improves insulin sensitivity in rodents and humans through a combination of metabolic actions, including partitioning of lipid stores and the regulation of metabolic and inflammatory mediators termed adipokines. PPARgamma signaling has also been implicated in the control of cell proliferation, atherosclerosis, macrophage function, and immunity. Here, we review recent advances in our understanding of the diverse biological actions of PPARgamma with an eye toward the expanding therapeutic potential of PPARgamma agonist drugs.
1,798 citations
TL;DR: The expected two-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected, and a mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
Abstract: Glycosyltransferases catalyze glycosidic bond formation using sugar donors containing a nucleoside phosphate or a lipid phosphate leaving group. Only two structural folds, GT-A and GT-B, have been identified for the nucleotide sugar-dependent enzymes, but other folds are now appearing for the soluble domains of lipid phosphosugar-dependent glycosyl transferases. Structural and kinetic studies have provided new insights. Inverting glycosyltransferases utilize a direct displacement S(N)2-like mechanism involving an enzymatic base catalyst. Leaving group departure in GT-A fold enzymes is typically facilitated via a coordinated divalent cation, whereas GT-B fold enzymes instead use positively charged side chains and/or hydroxyls and helix dipoles. The mechanism of retaining glycosyltransferases is less clear. The expected two-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected. A mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
1,601 citations
TL;DR: HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified.
Abstract: Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.
1,542 citations
TL;DR: Bacteria comprise an exceptionally accessible experimental system that has provided many of the answers to the remaining puzzles in an anaerobic world, and current research seeks to identify these.
Abstract: Life evolved in an anaerobic world; therefore, fundamental enzymatic mechanisms and biochemical pathways were refined and integrated into metabolism in the absence of any selective pressure to avoid reactivity with oxygen. After photosystem II appeared, environmental oxygen levels rose very slowly. During this time, microorganisms acquired oxygen tolerance by jettisoning enzymes that use glycyl radicals and exposed low-potential iron-sulfur clusters, which can be directly poisoned by oxygen. They also developed mechanisms to defend themselves against superoxide (O2−) and hydrogen peroxide, partially reduced oxygen species that are generated as inadvertent by-products of aerobic metabolism. Contemporary organisms have inherited both the vulnerabilities and the defenses of these ancestral microbes. Current research seeks to identify these, and bacteria comprise an exceptionally accessible experimental system that has provided many of the answers. This manuscript reviews recent developments and identifies re...
1,379 citations
TL;DR: Although technical in nature, these developments have important implications for the expanded use of optical tweezers in biochemical research and thus should be of general interest.
Abstract: It has been over 20 years since the pioneering work of Arthur Ashkin, and in the intervening years, the field of optical tweezers has grown tremendously. Optical tweezers are now being used in the investigation of an increasing number of biochemical and biophysical processes, from the basic mechanical properties of biological polymers to the multitude of molecular machines that drive the internal dynamics of the cell. Innovation, however, continues in all areas of instrumentation and technique, with much of this work focusing on the refinement of established methods and on the integration of this tool with other forms of single-molecule manipulation or detection. Although technical in nature, these developments have important implications for the expanded use of optical tweezers in biochemical research and thus should be of general interest. In this review, we address these recent advances and speculate on possible future developments.
1,062 citations
TL;DR: Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale as mentioned in this paper, and the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.
Abstract: Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.
1,051 citations
TL;DR: A unified energetic and kinematic framework in the Rosetta program allows a wide range of molecular modeling problems, from fibril structure prediction to RNA folding to the design of new protein interfaces, to be readily investigated and highlights areas for improvement.
Abstract: Advances over the past few years have begun to enable prediction and design of macromolecular structures at near-atomic accuracy. Progress has stemmed from the development of reasonably accurate and efficiently computed all-atom potential functions as well as effective conformational sampling strategies appropriate for searching a highly rugged energy landscape, both driven by feedback from structure prediction and design tests. A unified energetic and kinematic framework in the Rosetta program allows a wide range of molecular modeling problems, from fibril structure prediction to RNA folding to the design of new protein interfaces, to be readily investigated and highlights areas for improvement. The methodology enables the creation of novel molecules with useful functions and holds promise for accelerating experimental structural inference. Emerging connections to crystallographic phasing, NMR modeling, and lower-resolution approaches are described and critically assessed.
900 citations
TL;DR: In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.
Abstract: Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Forster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.
692 citations
TL;DR: This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
Abstract: About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
642 citations
TL;DR: How recent work in nonmammalian model organisms has revealed new insight into the genetics of DR and how the discovery of DR-specific transcription factors will advance the understanding of this phenomenon are discussed.
Abstract: Reducing food intake to induce undernutrition but not malnutrition extends the life spans of multiple species, ranging from single-celled organisms to mammals. This increase in longevity by dietary restriction (DR) is coupled to profound beneficial effects on age-related pathology. Historically, much of the work on DR has been undertaken using rodent models, and 70 years of research has revealed much about the physiological changes DR induces. However, little is known about the genetic pathways that regulate the DR response and whether or not they are conserved between species. Elucidating these pathways may facilitate the design of targeted pharmaceutical treatments for a range of age-related pathologies. Here, we discuss how recent work in nonmammalian model organisms has revealed new insight into the genetics of DR and how the discovery of DR-specific transcription factors will advance our understanding of this phenomenon.
600 citations
TL;DR: In this review, insights are provided into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p 53 function in tumors.
Abstract: The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.
TL;DR: Iron-sulfur (Fe/S) proteins are involved in a wide variety of cellular processes such as enzymatic reactions, respiration, cofactor biosynthesis, ribosome biogenesis, regulation of gene expression, and DNA-RNA metabolism.
Abstract: Iron-sulfur (Fe/S) proteins are involved in a wide variety of cellular processes such as enzymatic reactions, respiration, cofactor biosynthesis, ribosome biogenesis, regulation of gene expression, and DNA-RNA metabolism. Assembly of Fe/S clusters, small inorganic cofactors, is assisted by complex proteinaceous machineries, which use cysteine as a source of sulfur, combine it with iron to synthesize an Fe/S cluster on scaffold proteins, and finally incorporate the cluster into recipient apoproteins. In eukaryotes, such as yeast and human cells, more than 20 components are known that facilitate the maturation of Fe/S proteins in mitochondria, cytosol, and nucleus. These biogenesis components also perform crucial roles in other cellular pathways, e.g., in the regulation of iron homeostasis or the modification of tRNA. Numerous diseases including several neurodegenerative and hematological disorders have been associated with defects in Fe/S protein biogenesis, underlining the central importance of this proce...
TL;DR: This review considers different therapeutic strategies that have arisen from knowledge of CFTR structure and function as well as its biosynthetic processing, intracellular trafficking, and turnover.
Abstract: Mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel cause cystic fibrosis (CF). The multidomain integral membrane glycoprotein, a member of the adenine nucleotide-binding cassette (ABC) transporter family, conserved in metazoan salt-transporting tissues, is required to control ion and fluid homeostasis on epithelial surfaces. This review considers different therapeutic strategies that have arisen from knowledge of CFTR structure and function as well as its biosynthetic processing, intracellular trafficking, and turnover.
TL;DR: Findings obtained from genetically modified neurons and neuroendocrine cells, as well as from reconstituted systems, are summarized to reveal the molecular mechanism by which Ca(2+)-acting on the synaptic vesicle (SV) protein synaptotagmin I (syt)-triggers rapid exocytosis.
Abstract: Neurotransmitter release at synapses involves a highly specialized form of membrane fusion that is triggered by Ca(2+) ions and is optimized for speed. These observations were established decades ago, but only recently have the molecular mechanisms that underlie this process begun to come into view. Here, we summarize findings obtained from genetically modified neurons and neuroendocrine cells, as well as from reconstituted systems, which are beginning to reveal the molecular mechanism by which Ca(2+)-acting on the synaptic vesicle (SV) protein synaptotagmin I (syt)-triggers rapid exocytosis. This work sheds light not only on presynaptic aspects of synaptic transmission, but also on the fundamental problem of membrane fusion, which has remained a puzzle that has yet to be solved in any biological system.
TL;DR: The mammalian genome contains 10 enzymatically active secreted phospholipases A2 (sPLA2s) and two sPLA2-related proteins devoid of lipolytic enzymatic activity.
Abstract: Phospholipases A2 (PLA2s) are esterases that hydrolyze the sn-2 ester of glycerophospholipids and constitute one of the largest families of lipid hydrolyzing enzymes. The mammalian genome contains 10 enzymatically active secreted PLA2s (sPLA2s) and two sPLA2-related proteins devoid of lipolytic enzymatic activity. In addition to the well-established functions of one of these enzymes in digestion of dietary phospholipids and another in host defense against bacterial infections, accumulating evidence shows that some of these sPLA2s are involved in arachidonic acid release from cellular phospholipids for the biosynthesis of eicosanoids, especially during inflammation. More speculative results suggest the involvement of one or more sPLA2s in promoting atherosclerosis and cancer. In addition, the mammalian genome encodes several types of sPLA2-binding proteins, and mounting evidence shows that sPLA2s may have functions related to binding to cellular target proteins in a manner independent of their lipolytic enzymatic activity.
TL;DR: It is found that the energetics involving backbone hydrogen bonding controls shifts in protein stability almost entirely, with osmolyte cosolvents simply dialing between solvent- backbone versus backbone-backbone hydrogen bonds, as a function of solvent quality.
Abstract: We seek to understand the link between protein thermodynamics and protein structure in molecular detail. A classical approach to this problem involves assessing changes in protein stability resulting from added cosolvents. Under any given conditions, protein molecules in aqueous buffer are in equilibrium between unfolded and folded states, U(nfolded) � N(ative). Addition of organic osmolytes, small uncharged compounds found throughout nature, shift this equilibrium. Urea, a denaturing osmolyte, shifts the equilibrium toward U; trimethylamine N-oxide (TMAO), a protecting osmolyte, shifts the equilibrium toward N. Using the Tanford Transfer Model, the thermodynamic response to many such osmolytes has been dissected into groupwise free energy contributions. It is found that the energetics involving backbone hydrogen bonding controls these shifts in protein stability almost entirely, with osmolyte cosolvents simply dialing between solvent-backbone versus backbone-backbone hydrogen bonds, as a function of solvent quality. This reciprocal relationship establishes the essential link between protein thermodynamics and the protein’s hydrogen-bonded backbone structure.
TL;DR: It is concluded that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance the understanding of protein folding.
Abstract: Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the singlemolecule level have been developed. Advances in single-molecule fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.
TL;DR: The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins, which determine the specific cellular functions of the DNA ligase isozymes.
Abstract: DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.
TL;DR: Proteins belonging to the XPF/MUS81 family play important roles in the repair of DNA lesions caused by UV-light or DNA cross-linking agents and determining how they recognize specific DNA substrates and promote key repair reactions is an important challenge for the future.
Abstract: Proteins belonging to the XPF/MUS81 family play important roles in the repair of DNA lesions caused by UV-light or DNA cross-linking agents. Most eukaryotes have four family members that assemble into two distinct heterodimeric complexes, XPF-ERCC1 and MUS81-EME1. Each complex contains one catalytic and one noncatalytic subunit and exhibits endonuclease activity with a variety of 3'-flap or fork DNA structures. The catalytic subunits share a characteristic core containing an excision repair cross complementation group 4 (ERCC4) nuclease domain and a tandem helix-hairpin-helix (HhH)(2) domain. Diverged domains are present in the noncatalytic subunits and may be required for substrate targeting. Vertebrates possess two additional family members, FANCM and Fanconi anemia-associated protein 24 kDa (FAAP24), which possess inactive nuclease domains. Instead, FANCM contains a functional Superfamily 2 (SF2) helicase domain that is required for DNA translocation. Determining how these enzymes recognize specific DNA substrates and promote key repair reactions is an important challenge for the future.
TL;DR: Superb images of native membranes have been recorded, and a quantitative interpretation of the data acquired using the AFM tip has become possible, and multifunctional probes to simultaneously acquire information on the topography and electrical properties of membrane proteins have been produced.
Abstract: Evolution has tuned membrane proteins to exist in a lipid bilayer, provide for cell-cell communication, transport solutes, and convert energies. These proteins exhibit a hydrophobic belt that interacts with the lipid bilayer. Detergents are therefore used to extract membrane proteins and keep them in solution for purification and subsequent analyses. However, most membrane proteins are unstable when solubilized and hence often not accessible to NMR or X-ray crystallography. The atomic force microscope (AFM) is a powerful tool for imaging and manipulating membrane proteins in their native state. Superb images of native membranes have been recorded, and a quantitative interpretation of the data acquired using the AFM tip has become possible. In addition, multifunctional probes to simultaneously acquire information on the topography and electrical properties of membrane proteins have been produced. This progress is discussed here and fosters expectations for future developments and applications of AFM and single-molecule force spectroscopy.
TL;DR: An approach to integrate structural information gathered at multiple levels of the biological hierarchy--from atoms to cells--into a common framework is proposed, illustrated by determining the configuration of the 456 proteins in the nuclear pore complex from baker's yeast.
Abstract: To understand the cell, we need to determine the macromolecular assembly structures, which may consist of tens to hundreds of components. First, we review the varied experimental data that characterize the assemblies at several levels of resolution. We then describe computational methods for generating the structures using these data. To maximize completeness, resolution, accuracy, precision, and efficiency of the structure determination, a computational approach is required that uses spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. This approach is illustrated by determining the configuration of the 456 proteins in the nuclear pore complex (NPC) from baker's yeast. With these tools, we are poised to integrate structural information gathered at multiple levels of the biological hierarchy--from atoms to cells--into a common framework.
TL;DR: Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity.
Abstract: Single-molecule techniques have advanced our understanding of transcription by RNA polymerase (RNAP). A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps (OTs) have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriop...
TL;DR: The contributions of single-molecule experiments to providing answers to questions such as: How much energy is required to unfold a secondary or tertiary structure?
Abstract: Understanding how RNA folds and what causes it to unfold has become more important as knowledge of the diverse functions of RNA has increased. Here we review the contributions of single-molecule experiments to providing answers to questions such as: How much energy is required to unfold a secondary or tertiary structure? How fast is the process? How do helicases unwind double helices? Are the unwinding activities of RNA-dependent RNA polymerases and of ribosomes different from other helicases? We discuss the use of optical tweezers to monitor the unfolding activities of helicases, polymerases, and ribosomes, and to apply force to unfold RNAs directly. We also review the applications of fluorescence and fluorescence resonance energy transfer to measure RNA dynamics.
TL;DR: This review chronicles the key discoveries in this nascent field, which have demonstrated the power and promise of single-molecule techniques in the study of translation.
Abstract: Decades of studies have established translation as a multistep, multicomponent process that requires intricate communication to achieve high levels of speed, accuracy, and regulation. A crucial next step in understanding translation is to reveal the functional significance of the large-scale motions implied by static ribosome structures. This requires determining the trajectories, timescales, forces, and biochemical signals that underlie these dynamic conformational changes. Single-molecule methods have emerged as important tools for the characterization of motion in complex systems, including translation. In this review, we chronicle the key discoveries in this nascent field, which have demonstrated the power and promise of single-molecule techniques in the study of translation.
TL;DR: Some of the advantages of single-molecule methods over their bulk counterparts are discussed and it is argued that these advantages should help establish them as essential tools in the technical arsenal of the modern biochemist.
Abstract: It has been over one-and-a-half decades since methods of single-molecule detection and manipulation were first introduced in biochemical research. Since then, the application of these methods to an expanding variety of problems has grown at a vertiginous pace. While initially many of these experiments led more to confirmatory results than to new discoveries, today single-molecule methods are often the methods of choice to establish new mechanism-based results in biochemical research. Throughout this process, improvements in the sensitivity, versatility, and both spatial and temporal resolution of these techniques has occurred hand in hand with their applications. We discuss here some of the advantages of single-molecule methods over their bulk counterparts and argue that these advantages should help establish them as essential tools in the technical arsenal of the modern biochemist.
TL;DR: This review focuses on the use of structural information in combination with computational tools to predict new protein interactions, to determine which interactions are compatible with each other, to obtain some functional insight into single and multiple mutations, and to estimate equilibrium and kinetic parameters.
Abstract: Determining protein interaction networks and predicting network changes in time and space are crucial to understanding and modeling a biological system. In the past few years, the combination of experimental and computational tools has allowed great progress toward reaching this goal. Experimental methods include the large-scale determination of protein interactions using two-hybrid or pull-down analysis as well as proteomics. The latter one is especially valuable when changes in protein concentrations over time are recorded. Computational tools include methods to predict and validate protein interactions on the basis of structural information and bioinformatics tools that analyze and integrate data for the same purpose. In this review, we focus on the use of structural information in combination with computational tools to predict new protein interactions, to determine which interactions are compatible with each other, to obtain some functional insight into single and multiple mutations, and to estimate ...
TL;DR: A progress report toward a complete biomechanical understanding of a generalized bacterial cell is reviewed, with the goal of eventually integrating genetic, biochemical, imaging, and biophysical data into spatially explicit, mechanically predictive models of whole cells is highlighted.
Abstract: Following decades of research in genetics and biochemistry, the basic metabolism of bacteria is now well understood. In addition to core metabolism, however, bacterial cells also perform a number of mechanical tasks such as maintaining a characteristic shape, moving within their environment, segregating their genome, and dividing. Major advances in imaging technologies including fluorescence light microscopy (fLM) and electron cryotomography (ECT) have provided new insight into the bacterial ultrastructures that accomplish these tasks. It is now clear, for instance, that bacteria are highly organized, possessing cytoskeletons, specifically arranged genomes, internal compartments, and carefully positioned macromolecular machines. These structures and their functions are reviewed here in the form of a progress report toward a complete biomechanical understanding of a generalized bacterial cell. The goal of eventually integrating genetic, biochemical, imaging, and biophysical data into spatially explicit, mechanically predictive models of whole cells is highlighted.
TL;DR: An approach that may prove useful in analyzing aging is outlined, in which the function of the organism is described as a set of interacting physiological systems and the methods of metabolic control analysis are adapted to determine which modifications are important for aging.
Abstract: Aging is due to the accumulation of damage over time that affects the function and survival of the organism; however, it has proven difficult to infer the relative importance of the many processes that contribute to aging. To address this, here we outline an approach that may prove useful in analyzing aging. In this approach, the function of the organism is described as a set of interacting physiological systems. Degradation of their outputs leads to functional decline and death as a result of aging. In turn, degradation of the system outputs is attributable to changes at the next hierarchical level down, the cell, through changes in cell number or function, which are in turn a consequence of the metabolic history of the cell. Within this framework, we then adapt the methods of metabolic control analysis (MCA) to determine which modifications are important for aging. This combination of a hierarchical framework and the methodologies of MCA may prove useful both for thinking about aging and for analyzing it experimentally.
TL;DR: This review presents several methods of determining complex chemical reaction mechanisms and their functions based on correlation functions of measured time series of concentrations of chemical species, and one method is based on measurements of temporal responses of concentrations to various perturbations of arbitrary magnitude.
Abstract: This review presents several methods of determining complex chemical reaction mechanisms and their functions. One method is based on correlation functions of measured time series of concentrations of chemical species, another is on measurements of temporal responses of concentrations to various perturbations of arbitrary magnitude, the third deals with the analysis of oscillatory systems, and the fourth describes the use of genetic algorithms. All methods are applicable to chemical, biochemical, and biological reaction systems and to genetic networks. The methods depend on the design of appropriate experiments for the whole system and corresponding theories for interpretation that lead to information on the causal chemical connectivity of species, reaction pathways, reaction mechanisms, control centers in the system, and functions of the system. The first three methods require no assumption of a model or hypothesis, nor extensive calculations, unlike the interpretation of measurements made on a gene network at only one time. The methods offer advantageous approaches to systems biology.
TL;DR: The regulatory GTPase cycle is presented, for the first time, an explicit mechanism for receptor action that predicts that two mechanisms could account for stimulation of adenylyl cyclase activity-either by the familiar hormone stimulation of the activation reaction or by an inhibition of the turn-off reaction.
Abstract: The mechanism of transmembrane signaling by the receptor-activated adenylyl cyclase was an enigma. It was suggested that hydrolysis of GTP is a turn-off mechanism that resets the active adenylyl cyclase to the inactive state. To test this hypothesis, we developed a specific GTPase assay and found that the catecholamine adrenergic agonists stimulated the hydrolysis of GTP. To resolve the question of how the hormone concurrently stimulates GTP hydrolysis and activates the adenylyl cyclase, we suggested the regulatory GTPase cycle. Thus, because the hormone facilitates the binding of GTP, which is subsequently hydrolyzed, the regulatory cycle results in a hormone-stimulated GTPase activity. This model also predicts that two mechanisms could account for stimulation of adenylyl cyclase activity-either by the familiar hormone stimulation of the activation reaction or by an inhibition of the turn-off reaction. Indeed, we showed that cholera toxin enhances adenylyl cyclase activity by inhibition of GTP hydrolysis. Finally, we also showed that the hormone-activated receptor stimulates adenylyl cyclase activity by facilitating the exchange of bound GDP for free GTP. Thus, we presented, for the first time, an explicit mechanism for receptor action.