From steroid receptors to cytokines: the thermodynamics of self-associating systems

Since 1987, the Gibbs Conference on Biothermodynamics has maintained a focus on understanding the quantitative aspects of gene regulatory systems. These studies coupled rigorous techniques with exact theory to dissect the linked reactions associated with bacterial and lower eukaryotic gene regulation. However, only in the last ten years has it become possible to apply this approach to clinically relevant, human gene regulatory systems. Here we summarize our work on the thermodynamics of human steroid receptors and their interactions with multi-site promoter sequences, highlighting results not available from more traditional biochemical and structural approaches. Noting that the Gibbs Conference has also served as a vehicle to promote the broader use of thermodynamics in understanding biology, we then discuss collaborative work on the hydrodynamics of a cytokine implicated in tumor suppression, prostate derived factor (PDF).     

The use of analytical sedimentation velocity to extract thermodynamic linkage

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS.     

PML nuclear bodies and chromatin dynamics: catch me if you can!

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.     

Exploring and exploiting allostery: Models, evolution, and drug targeting

The concept of allostery was elaborated almost 50years ago by Monod and coworkers to provide a framework for interpreting experimental studies on the regulation of protein function. In essence, binding of a ligand at an allosteric site affects the function at a distant site exploiting protein flexibility and reshaping protein energy landscape. Both monomeric and oligomeric proteins can be allosteric. In the past decades, the behavior of allosteric systems has been analyzed in many investigations while general theoretical models and variations thereof have been steadily proposed to interpret the experimental data. Allostery has been established as a fundamental mechanism of regulation in all organisms, governing a variety of processes that range from metabolic control to receptor function and from ligand transport to cell motility. A number of studies have shed light on how evolutionary pressures have favored and molded the development of allosteric features in specific macromolecular systems. The widespread occurrence of allostery has been recently exploited for the development and design of allosteric drugs that bind to either physiological or non-physiological allosteric sites leading to gain of function or loss of function. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Fungal proteolytic enzymes: Features of the extracellular proteases of xylotrophic basidiomycetes

Fungal proteolytic enzymes attract the attention of researches due to such features as high diversity, broad substrate specificity, and stability under extreme conditions. Their functional role is also interesting; it includes a number of processes from the hydrolysis of macromolecular substrates under extremely low nitrogen content to initiation and maintenance of pathogenesis. In the present review, the features of the extracellular proteases of xylotrophic basidiomycetes are discussed. This group is important for the functioning of biological communities and participates in the biological destruction of plant debris; moreover, they are widely used as a source of nutrients and medicines. The review stresses the issues of classification of fungal proteases, their biochemical characteristics and physiological role, as well as the regulation of their activity in the course of fungal growth.     

Simple primary structure, complex turnover regulation and multiple roles of hyaluronan

Hyaluronan is a major macromolecular polysaccharide component of the extra-cellular matrix that confers structural frameworks for cells. Despite its relatively simple chemical composition, hyaluronan mediates many other important functional aspects including signalling activity during embryonic morphogenesis, cellular regeneration and wound healing. Abnormalities in hyaluronan metabolism have been implicated in many diseases, such as inflammatory disorders, cardiovascular diseases and cancer. To date, it has become increasingly clear that hyaluronan production in vertebrates is tightly regulated by three hyaluronan synthases and that hyaluronan catabolism is regulated by an enzymatic degradation reaction involving several hyaluronidases. Together, these discoveries have provided key insights into the physiological roles of hyaluronan and a deeper understanding of the mechanisms underlying altered hyaluronan turnover in diseases. The central aim of this review article is therefore to highlight the multiple roles of hyaluronan in physiological and pathological states via its complex turnover regulation.     

Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli

With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.     

Polyhedral organelles compartmenting bacterial metabolic processes

Bacterial polyhedral organelles are extremely large macromolecular complexes consisting of metabolic enzymes encased within a multiprotein shell that is somewhat reminiscent of a viral capsid. Recent investigations suggest that polyhedral organelles are widely used by bacteria for optimizing metabolic processes. The distribution and diversity of these unique structures has been underestimated because many are not formed during growth on standard laboratory media and because electron microscopy is required for their observation. However, recent physiological studies and genomic analyses tentatively indicate seven functionally distinct organelles distributed among over 40 genera of bacteria. Functional studies conducted thus far are consistent with the idea that polyhedral organelles act as microcompartments that enhance metabolic processes by selectively concentrating specific metabolites. Relatively little is known about how this is achieved at the molecular level. Possible mechanisms include regulation of enzyme activity or efficiency, substrate channeling, a selectively permeable protein shell, and/or differential solubility of metabolites within the organelle. Given their complexity and distinctive structure, it would not be surprising if aspects of their biochemical mechanism are unique. Therefore, the unusual structure of polyhedral organelles raises intriguing questions about their assembly, turnover, and molecular evolution, very little of which is understood.     

Sodium/calcium exchanger (NCX1) macromolecular complex

The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.     

Introduction to the Rosetta Special Collection

The Rosetta macromolecular modeling software is a versatile, rapidly developing set of tools that are now being routinely utilized to address state-of-the-art research challenges in academia and industrial research settings. A Rosetta Conference (RosettaCon) describing updates to the Rosetta source code is held annually. Every two years, a Rosetta Conference (RosettaCon) special collection describing the results presented at the annual conference by participating RosettaCommons labs is published by the Public Library of Science (PLOS). This is the introduction to the third RosettaCon 2014 Special Collection published by PLOS.     

Physiological importance of RNA and protein mobility in the cell nucleus

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways.     

Under Construction: Building the Macromolecular Superstructure and Signaling Components of an Electrical Synapse

A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36-containing electrical synapses in brain--specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.     

The role of polyamines in animal cell physiology

The ubiquitous distribution of polyamines in nature suggests that they fulfil some fundamental role(s) in living organisms. In animal cells, polyamine content closely parallels changes in the rate of cell proliferation so that the highest content is always observed in rapidly growing cells. The activity of ornithine decarboxylase (which is the first enzyme in the polyamine biosynthetic pathway) has been found to increase significantly in many systems shortly after exposure to hormones. Also, addition of polyamines greatly stimulates cell-free macromolecular synthesis. Observations such as these have suggested that polyamine accumulation stimulates cell growth and is important in the regulation of macromolecular biosynthesis. However, it is also possible to interpret such data as evidence that polyamine accumulation is the result, not the cause, of increased cell growth. This review supports the latter concept and re-examines the significance of the early induction of ornithine decarboxylase activity and of the stimulatory effects of exogenous polyamine on macromolecular synthesis. It is proposed that the polyamines are important only in maintaining cell growth that has already been stimulated by other factors and that their biosynthesis is to a large extent determined by the accumulation of RNA in the cell.     

NO在植物生长发育和环境胁迫响应中的作用

一氧化氮(NO)是具有生物活性和信号转导作用的气体活性分子,它不仅对植物的许多生命活动如种子萌发、生长和衰老等具有直接的生理调节功能,而且作为防御反应中的关键信使,参与了植物对外界环境胁迫的响应,如干旱胁迫、热胁迫、盐胁迫、UV-B辐射、臭氧胁迫、重金属胁迫、机械损伤以及植物抗病反应。NO与各种激素如乙烯、脱落酸、水杨酸、生长素和细胞分裂素等,在调节植物的生理活动与信号转导方面有明显的协同作用,通过激素起作用可能是植物内源NO作用的机理之一。探明在正常生长状况下植物内源NO对植物生长发育的调控机制及其参与信号转导的生理机制是目前研究的重点。     

Growing Out of Stress: The Role of Cell- and Organ-Scale Growth Control in Plant Water-Stress Responses

Water is the most limiting resource on land for plant growth, and its uptake by plants is affected by many abiotic stresses, such as salinity, cold, heat, and drought. While much research has focused on exploring the molecular mechanisms underlying the cellular signaling events governing water-stress responses, it is also important to consider the role organismal structure plays as a context for such responses. The regulation of growth in plants occurs at two spatial scales: the cell and the organ. In this review, we focus on how the regulation of growth at these different spatial scales enables plants to acclimate to water-deficit stress. The cell wall is discussed with respect to how the physical properties of this structure affect water loss and how regulatory mechanisms that affect wall extensibility maintain growth under water deficit. At a higher spatial scale, the architecture of the root system represents a highly dynamic physical network that facilitates access of the plant to a heterogeneous distribution of water in soil. We discuss the role differential growth plays in shaping the structure of this system and the physiological implications of such changes.