Globular Subunits Research Articles

A biofunctional review of C-reactive protein (CRP) as a mediator of inflammatory and immune responses: differentiating pentameric and modified CRP isoform effects.

C-reactive protein (CRP) is an acute phase, predominantly hepatically synthesized protein, secreted in response to cytokine signaling at sites of tissue injury or infection with the physiological function of acute pro-inflammatory response. Historically, CRP has been classified as a mediator of the innate immune system, acting as a pattern recognition receptor for phosphocholine-containing ligands. For decades, CRP was envisioned as a single, non-glycosylated, multi-subunit protein arranged non-covalently in cyclic symmetry around a central void. Over the past few years, however, CRP has been shown to exist in at least three distinct isoforms: 1.) a pentamer of five identical globular subunits (pCRP), 2.) a modified monomer (mCRP) resulting from a conformational change when subunits are dissociated from the pentamer, and 3.) a transitional isoform where the pentamer remains intact but is partially changed to express mCRP structural characteristics (referred to as pCRP* or mCRPm). The conversion of pCRP into mCRP can occur spontaneously and is observed under commonly used experimental conditions. In careful consideration of experimental design used in published reports of in vitro pro- and anti-inflammatory CRP bioactivities, we herein provide an interpretation of how distinctive CRP isoforms may have affected reported results. We argue that pro-inflammatory amplification mechanisms are consistent with the biofunction of mCRP, while weak anti-inflammatory mechanisms are consistent with pCRP. The interplay of each CRP isoform with specific immune cells (platelets, neutrophils, monocytes, endothelial cells, natural killer cells) and mechanisms of the innate immune system (complement), as well as differences in mCRP and pCRP ligand recognition and effector functions are discussed. This review will serve as a revised understanding of the structure-function relationship between CRP isoforms as related to inflammation and innate immunity mechanisms.

The gene order in the nuo-operon is not essential for the assembly of E. coli complex I

Energy-converting NADH: ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular energy metabolism. Bacterial complex I is generally composed of 14 different subunits, seven of which are membranous and the other seven are globular proteins. They are encoded by the nuo-operon, whose gene order is strictly conserved in bacteria. The operon starts with nuoA encoding a membranous subunit followed by genes encoding globular subunits. To test the idea that NuoA acts as a seed to initiate the assembly of the complex in the membrane, we generated mutants that either lacked nuoA or contain nuoA at a different position within the operon. To enable the detection of putative assembly intermediates, the globular subunit NuoF and the membranous subunit NuoM were individually decorated with the fluorescent protein mCherry. Deletion of nuoA led to the assembly of an inactive complex in the membrane containing NuoF and NuoM. Re-arrangement of nuoA within the nuo-operon led to a slightly diminished amount of complex I in the membrane that was fully active. Thus, nuoA but not its distinct position in the operon is required for the assembly of E. coli complex I. Furthermore, we detected a previously unknown assembly intermediate in the membrane containing NuoM that is present in greater amounts than complex I.

Nonequilibrium self-assembly of multiple stored targets in a dimer-based system.

Nonequilibrium self-assembly can be found in various biological processes where chemical potential gradients are exploited to steer the system to a desired organized structure with a particular function. Microtubules, for example, are composed of two globular protein subunits, α-tubulin and β-tubulin, which bind together to form polar dimers that self-assemble a hollow cylinder structure in a process driven by GTPase activity. Inspired by this process, we define a generic self-assembly lattice model containing particles of two subunits, which is driven out-of-equilibrium by a dimer-favoring local driving force. Using Monte Carlo simulations, we characterize the ability of this system to restore pre-encoded target structures as a function of the initial seed size, interaction energy, chemical potential, number of target structures, and strength of the nonequilibrium drive. We demonstrate some intriguing consequences of the drive, such as a smaller critical seed and an improved target assembly stability, compared to the equilibrium scenario. Our results can expand the theoretical basis of nonequilibrium self-assembly and provide deeper understanding of how nonequilibrium driving can overcome equilibrium constraints.

C-Reactive Protein and Cancer: Interpreting the Differential Bioactivities of Its Pentameric and Monomeric, Modified Isoforms.

C-reactive protein (CRP) was first recognized in the 1940s as a protein that appeared in blood during acute episodes of infectious disease. Its presence and pharmacodynamics were found in essentially all diseases that involved tissue damage and inflammation. Identified as a major component of the innate, unlearned immunity, it became a useful diagnostic marker for the extent of inflammation during disease exacerbation or remission. Efforts to define its true biological role has eluded clear definition for over a half-century. Herein, a unifying concept is presented that explains both pro-inflammatory and anti-inflammatory activities of CRP. This concept involves the recognition and understanding that CRP can be induced to undergo a pronounced, non-proteolytic reorganization of its higher-level protein structures into conformationally distinct isomers with distinctive functional activities. This process occurs when the non-covalently associated globular subunits of the pentameric isoform (“pCRP”) are induced to dissociate into a monomeric isoform (“mCRP”). mCRP consistently and potently provides pro-inflammatory activation and amplification activities. pCRP provides weak anti-inflammatory activities consistent with low-level chronic inflammation. mCRP can spontaneously form in purified pCRP reagents in ways that are not immediately recognized during purification and certification analyses. By now understanding the factors that influence pCRP dissociate into mCRP, many published reports investigating CRP as a biological response modifier of host defense can be reevaluated to include a discussion of how each CRP isoform may have affected the generated results. Specific attention is given to in vitro and in vivo studies of CRP as an anti-cancer agent.

Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please.

Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular “antennae” in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.

Intra-subunit Disulfide Determines the Conversion and Structural Stability of CRP Isoforms.

C-reactive protein (CRP) is a major human acute-phase reactant that is composed of five identical subunits. CRP dissociates into subunits at inflammatory loci forming monomeric CRP (mCRP) with substantially enhanced activities, which can be further activated by reducing the intra-subunit disulfide bond. However, conformational changes underlying the activation process of CRP are less well understood. Conformational changes accompanying the conversion of CRP to mCRP with or without reduction were examined with circular dichroism spectroscopy, fluorescence spectroscopy, electron microscopy, size-exclusion chromatography, and neoepitope expression. The conversion of CRP to mCRP follows a two-stage process. In the first stage, CRP dissociates into molten globular subunits characterized by intact secondary structure elements with greatly impaired tertiary packing. In the second stage, these intermediates completely lose their native subunit conformation and assemble into high-order aggregates. The inclusion of reductant accelerates the formation of molten globular subunits in the first step and promotes the formation of more compact aggregates in the second stage. We further show a significant contribution of electrostatic interactions to the stabilization of native CRP. The conformational features of dissociated subunits and the aggregation of mCRP may have a key impact on their activities.

C - REACTIVE PROTEIN: AN INFLAMMATORY BIOMARKER PRESENT IN MULTIPLE DISEASE PATHOGENESIS

C-reactive protein (CRP) is an acute-phase protein belongs to pentraxins family with a feature of ve identical globular subunits. Two different isoforms i.e. pentameric CRP (pCRP) and monomeric CRP (mCRP) have different key role in regulation. The key biological function of CRP is host defense using classical complement pathway against bacterial infection and involved in removal of apoptotic and necrotic cells. Although CRP is mainly associated with inammation in bacterial infection such as tuberculosis or Pneumococcus infection, however it is also presence in atherosclerosis, cardiovascular disease (CVD) and in various cancers which make it good candidate for prognostic study in different clinical conditions.

Regulation of C-reactive protein conformation in inflammation.

C-reactive protein (CRP) is a non-specific diagnostic marker of inflammation and an evolutionarily conserved protein with roles in innate immune signaling. Natural CRP is composed of five identical globular subunits that form a pentamer, but the role of pentameric CRP (pCRP) during inflammatory pathogenesis remains controversial. Emerging evidence suggests that pCRP can be dissociated into monomeric CRP (mCRP) that has major roles in host defenses and inflammation. Here, we discuss our current knowledge of the dissociation mechanisms of pCRP and summarize the stepwise conformational transition model to mCRP to elucidate how CRP dissociation contributes to proinflammatory activity. These discussions will evoke new understanding of this ancient protein.

The hierarchical emergence of worm-like chain behaviour from globular domain polymer chains.

Biological organisms make use of hierarchically organised structures to modulate mechanical behaviour across multiple lengthscales, allowing microscopic objects to generate macroscopic effects. Within these structural hierarchies, the resultant physical behaviour of the entire system is determined not only by the intrinsic mechanical properties of constituent subunits, but also by their organisation in three-dimensional space. When these subunits are polyproteins, colloidal chains or other globular domain polymers, the Kratky-Porod model is often assumed for the individual subunits. Hence, it is implicitly asserted that the polymeric object has an intrinsic parameter, the persistence length, that defines its flexibility. However, the persistence lengths extracted from experiment vary, and are often relatively small. Through a series of simulations on polymer chains formed of globular subunits, we show that the persistence length itself is a hierarchical structural property, related not only to the intrinsic mechanical properties of the underlying monomeric subunits, but emerging due to the organisation of inhomogenous geometry along the polymer contour.

Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

Actin filaments are a major component of the cytoskeleton involved in many basic processes such as cell migration, cell adhesion, cell division and muscle contraction. The most important property of actin is that it forms filaments (F-actin) from globular subunits (G-actin). In the 1960s, Oosawa and co-workers developed a model for the G- to F-actin transition where the rate-limiting step is the formation of a stable actin nucleus, later determined to consist of 2-4 actin monomers. Nuclei are elongated by the addition of monomers to the filament ends. This model is widely accepted, although the molecular details could never be visualised experimentally. Here, we used interferometric scattering microscopy (iSCAT) to monitor nucleation and elongation of actin filaments at the single-molecule level. Building on recent improvements in label-free single molecule sensitivity and mass accuracy, we could reveal the polydispersity of G-actin at concentrations relevant to nucleation and filament growth, showing signatures of small protein oligomers in line with the nuclei sizes discussed above. Using the same approach, we could monitor the nanoscopic arrival of individual subunits dynamically attaching to and detaching from the filament tip during polymerisation. Contrary to the standard model, where actin grows exclusively from monomers, we also found signatures of larger oligomers being added to the filament. Together with our polydispersity measurements, these results point towards a nucleation and growth mechanism for actin filaments based on monomers and small oligomers, rather than exclusively monomers. These results demonstrate the potential of iSCAT for label-free single-molecule imaging and for investigating the mechanisms of mesoscopic dynamics in solution.

Low-dimensional manifold of actin polymerization dynamics

Actin filaments are critical components of the eukaryotic cytoskeleton, playing important roles in a number of cellular functions, such as cell migration, organelle transport, and mechanosensation. They are helical polymers with a well-defined polarity, composed of globular subunits that bind nucleotides in one of three hydrolysis states (ATP, ADP-Pi, or ADP). Mean-field models of the dynamics of actin polymerization have succeeded in, among other things, determining the nucleotide profile of an average filament and resolving the mechanisms of accessory proteins. However, these models require numerical solution of a high-dimensional system of nonlinear ordinary differential equations. By truncating a set of recursion equations, the Brooks–Carlsson (BC) model reduces dimensionality to 11, but it still remains nonlinear and does not admit an analytical solution, hence, significantly hindering understanding of its resulting dynamics. In this work, by taking advantage of the fast timescales of the hydrolysis states of the filament tips, we propose two model reduction schemes: the quasi steady-state approximation model is five-dimensional and nonlinear, whereas the constant tip (CT) model is five-dimensional and linear, resulting from the approximation that the tip states are not dynamic variables. We provide an exact solution of the CT model and use it to shed light on the dynamical behaviors of the full BC model, highlighting the relative ordering of the timescales of various collective processes, and explaining some unusual dependence of the steady-state behavior on initial conditions.

In vitro gastrointestinal digestion of pea protein isolate as a function of pH, food matrices, autoclaving, high-pressure and re-heat treatments

This study investigated the influence of pH and processing conditions (autoclave at 93 °C/13 min or high pressure processing (HPP) at 600 MPa/5 min without/with follow-up reheating at 80 °C/30 min) on the digestibility of pea protein isolate. Both aqueous solutions and real food matrices (apple and carrot purees) containing pea protein was examined at 37 °C. In vitro gastrointestinal digestion was followed using sodium dodecyl sulphate polyacrylamide gel electrophoresis, titrimetric techniques and theoretical calculations. Pea protein with HPP followed by re-heating showed the highest rate of proteolysis in gastric conditions. In case of sequential intestinal digestion of the gastric chyme, pea protein at pH 6.2 demonstrated higher degree and rate of digestibility as compared to that at pH 3.6, the latter being close to the isoelectric point of pea protein. However, autoclave treatments overshadowed such pH effects. Processing-induced enhancement in digestibility might be attributed to the unfolding of the globular pea protein subunits. Pea protein in the carrot puree was more digestible than in the apple puree, due to apple procyanidins binding to pea protein. These new findings might have important implications in designing the process parameters and selection of appropriate food matrices for delivering pea protein.

Dynamic Cylindrical Assembly of Triblock Copolymers by a Hierarchical Process of Covalent and Supramolecular Interactions

We have developed a hierarchical process that combines linear triblock copolymers into concentric globular subunits through strong chemical bonds and is followed by their supramolecular assembly via weak noncovalent interactions to afford one-dimensionally assembled, dynamic cylindrical nanostructures. The molecular brush architecture forces triblock copolymers to adopt intramolecular interactions within confined frameworks and then drives their intermolecular interactions in the mixtures of organic solvent and water. In contrast, the triblock copolymers, when not preconnected into the molecular brush architectures, organize only into globular assemblies.

Positioning of the HA Globular Head within the TLR5 Agonist, Flagellin, Modulates the Immunogenicity and Reactogenicity of a Novel-H1 Pandemic Influenza Vaccine (136.13)

Abstract The recent appearance of a novel H1N1 (nH1N1) virus has highlighted the importance of rapid response to emerging pandemic threats. VaxInnate has developed an influenza vaccine production system that permits rapid response on a global scale. The vaccine comprises the protective globular head subunit of HA fused covalently to the TLR 5 ligand flagellin. Using animal models that have successfully predicted the tolerated dose range in humans we have identified an optimal position for the HA globular head. Three constructs evaluated the globular head fused to the C terminus of flagellin (C-term), in place of domain 3 (R3) and in both positions (R3.2x). The three formats were tested in mouse immunogenicity and rabbit reactogenicity models. HAI assays were performed using CA/07/2009 virus. Rabbits were monitored for body temperature, food consumption and CRP. In mice, HAI titers >1:40 were achieved with 1 μg doses of all vaccines, but R3 and R3.2x vaccines yielded higher titers. In rabbits, the C-term vaccine elicited elevated temperature and CRP levels and reduced food consumption at doses well below R3 and R3.2x. Replacement of domain 3 of flagellin with the HA globular head, either R3 or R3.2x, proves safe and immunogenic in mice and rabbits. Our clinical experience with other flagellin-linked vaccines has correlated well with the rabbit model. The results predict that the R3 or R3.2x nH1 vaccines would be both safe and efficacious in humans.

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Solution characterization of [methyl- 13C]methionine HIV-1 reverse transcriptase by NMR spectroscopy

HIV reverse transcriptase (RT) is a primary target for drug intervention in the treatment of AIDS. We report the first solution NMR studies of [methyl- 13C]methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-nucleoside RT inhibitor (NNRTI) nevirapine. The selection of methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site methionine residues, M184 66 and M230 66. Observation of the M184 resonance is subunit dependent; in the p66 subunit the solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M184 51 resonance is shifted and broadened as M184 becomes buried in the protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions gives reasonable agreement for M184 66, while M230 66, located on the β-hairpin “primer grip”, is more mobile and solvent-exposed than suggested by crystal structures of the apo enzyme which have a “closed” fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the nevirapine, both the M184 66 and M230 66 resonances are significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Conformational features that might explain the multiple peaks are discussed.

Role of DNA methylation and histone modifications in structural maintenance of heterochromatin domains (chromocenters)

The effects of DNA methylation inhibitor 5-azacytidine (5-aza-C) and histone acetylation inhibitor trichostatine A (TSA) on the structure of pericentric heterochromatin in cultured mouse cells (L929) has been studied. After 48 h of 5-aza-C treatment, about 85% of the cells demonstrate transformation of chromocenters from ovoid to elongated structures. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules, well distinguishable by light microscopy. The same globular units can be revealed in hypotonic-treated control cells. 48 h of TSA treatment causes dramatic decrease in HP 1alpha content in the cells. Chromocenters in 25% of treated cells became highly decondenced and can not be reliably detected by light and electron microscopy. 85% of cells demonstrate globular chromocenters with low HP 1alpha content. Hypotonic treatment causes transformation of compact chromocenters into ring-like structures, which can be either single or clustered. Rings are formed by uniform fiber, in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the roles of epigenetic marks in them.

High‐resolution cryo‐SEM allows direct identification of F‐actin at the inner nuclear membrane of Xenopus oocytes by virtue of its structural features

The nuclear envelope of Xenopus laevis stage VI oocytes was studied in a high-resolution field emission cryo-scanning electron microscope to compare the level of structural preservation obtainable by different procedures of specimen preparation. All approaches generally allowed frequent detection of long filaments of about 10 nm in diameter that were attached to the nuclear envelope's inner membrane facing the nuclear interior. Structural details of these 10-nm filaments, however, could not be unveiled by standard procedures of specimen preparation and analysis, including critical point drying and imaging at room temperature. In contrast, after freeze-drying and imaging at -100 degrees C, the 10-nm filament type was found to be composed of distinct globular subunits of approximately 5 nm in diameter that were arranged in a helical manner with right-handed periodicity. Stereoscopic images showed that some of these filaments were lying directly on the membrane whereas others appeared to hover at a certain distance above the nuclear envelope. The appearance of these filaments was highly similar to that of in vitro polymerized F-actin analysed in parallel, and closely resembled the structural characteristics of F-actin filaments described earlier. By virtue of their structural features we therefore conclude that these filaments at the nuclear periphery represent F-actin. The high level of structural resolution obtainable by field emission cryo-SEM illustrates the potential of this method for studying details of biological structures in a subcellular context.

Isolation and partial characterisation of four novel plasma lectins from the American lobster Homarus americanus

Although numerous haemolymph-derived crustacean lectins are described, few have been reported for the American lobster Homarus americanus. In the present study, affinity chromatography was used to isolate and partially describe the carbohydrate affinity of four new lectins from H. americanus plasma. HaMBP and HaDNABP were homodimers of ∼30 kDa subunits which bound to mannan- and DNA-agarose columns, respectively. These proteins had partially overlapping elution profiles, and both shared and unique amino acid sequences and fragmentation patterns after trypsin digestion. A third homodimer of ∼29 kDa subunits eluted with HaMBP and HaDNABP under certain conditions. HaNBP occurred as a monomer and dimer of ∼40 kDa subunits and was recovered in relatively large quantities from mannan-agarose with N-acetylated sugars. Transmission electron microscopy revealed HaNBP to be a linear protein composed of multiple globular subunits.

Structural Analysis of the Saf Pilus by Electron Microscopy and Image Processing

Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a “beads on a string”-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.