Covalent modification of proteins as a threshold mechanism in development

Thresholds are a central but somewhat neglected aspect of cellular processes in development. An analysis has been made of the conditions in which different thresholds can be generated in the covalent modification of a number of target proteins when the concentration of an effector is continuously increased. It is assumed that the effector, which could represent a morphogen, activates, for example, kinases that phosphorylate the proteins. Thresholds are found when the modifying enzymes are saturated by their protein substrates, i.e. in conditions of zero-order ultrasensitivity (Goldbeter, A. & Koshland, D. E. 1981. An amplified sensitivity arising from covalent modification in biological systems. Proc. natn. Acad. Sci. U.S.A. 78, 6840-6844). Sequential thresholds can be generated when the kinase/phosphatase pairs differ either in the ratio of maximum modification rates or in the affinity of the effector for each kinase.     

Reciprocal Regulation as a Source of Ultrasensitivity in Two-Component Systems with a Bifunctional Sensor Kinase

Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations – a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme''s phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness – consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase''s phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two-component systems, but may apply more generally to bifunctional enzyme systems.     

Sensitivity and Robustness in Covalent Modification Cycles with a Bifunctional Converter Enzyme

Regulation by covalent modification is a common mechanism to transmit signals in biological systems. The modifying reactions are catalyzed either by two distinct converter enzymes or by a single bifunctional enzyme (which may employ either one or two catalytic sites for its opposing activities). The reason for this diversification is unclear, but contemporary theoretical models predict that systems with distinct converter enzymes can exhibit enhanced sensitivity to input signals whereas bifunctional enzymes with two catalytic sites are believed to generate robustness against variations in system’s components. However, experiments indicate that bifunctional enzymes can also exhibit enhanced sensitivity due to the zero-order effect, raising the question whether both phenomena could be understood within a common mechanistic model. Here, I argue that this is, indeed, the case. Specifically, I show that bifunctional enzymes with two catalytic sites can exhibit both ultrasensitivity and concentration robustness, depending on the kinetic operating regime of the enzyme’s opposing activities. The model predictions are discussed in the context of experimental observations of ultrasensitivity and concentration robustness in the uridylylation cycle of the PII protein, and in the phosphorylation cycle of the isocitrate dehydrogenase, respectively.     

Sensitivity and Robustness in Covalent Modification Cycles with a Bifunctional Converter Enzyme

Regulation by covalent modification is a common mechanism to transmit signals in biological systems. The modifying reactions are catalyzed either by two distinct converter enzymes or by a single bifunctional enzyme (which may employ either one or two catalytic sites for its opposing activities). The reason for this diversification is unclear, but contemporary theoretical models predict that systems with distinct converter enzymes can exhibit enhanced sensitivity to input signals whereas bifunctional enzymes with two catalytic sites are believed to generate robustness against variations in system’s components. However, experiments indicate that bifunctional enzymes can also exhibit enhanced sensitivity due to the zero-order effect, raising the question whether both phenomena could be understood within a common mechanistic model. Here, I argue that this is, indeed, the case. Specifically, I show that bifunctional enzymes with two catalytic sites can exhibit both ultrasensitivity and concentration robustness, depending on the kinetic operating regime of the enzyme’s opposing activities. The model predictions are discussed in the context of experimental observations of ultrasensitivity and concentration robustness in the uridylylation cycle of the PII protein, and in the phosphorylation cycle of the isocitrate dehydrogenase, respectively.     

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.     

Regulation through phosphorylation/dephosphorylation cascade systems

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.     

Carboxyl-terminal extensions beyond the conserved pentapeptide reduce rates of chemoreceptor adaptational modification

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In the well-characterized chemosensory systems of Escherichia coli and Salmonella spp., efficient modification by either enzyme is dependent on a conserved pentapeptide sequence, NWETF or NWESF, present at the extreme carboxyl terminus of high-abundance chemoreceptors. To what extent is position at the extreme carboxyl terminus important for pentapeptide-mediated enhancement of adaptational modification? Is this position equally important for enhancement of both enzyme activities? To address these questions, we created forms of high-abundance receptor Tsr or Tar carrying one, six, or eight additional amino acids extending beyond the pentapeptide at their carboxyl termini and assayed methylation, demethylation, deamidation, and ability to mediate chemotaxis. In vitro and in vivo, all three carboxyl-terminal extensions reduced pentapeptide-mediated enhancement of rates of adaptational modification. CheB-catalyzed reactions were more affected than CheR-catalyzed reactions. Effects were less severe for the complete sensory system in vivo than for the minimal system of receptor and modification enzymes in vitro. Notably, extended receptors mediated chemotaxis as efficiently as wild-type receptors, providing a striking example of robustness in chemotactic systems. This could reflect compensatory reductions of rates for both modification reactions, mitigation of effects of slower reactions by the intertwined circuitry of signaling and adaptation, or tolerance of a range of reactions rates for adaptational modification. No matter what the mechanism, the observations provide a challenging test for mathematical models of chemotaxis.     

Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive

Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades. Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems. Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient. We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme. This robust response of GS adenylylation is also observed for change in system parameters. From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure. By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status.     

Studies toward stereoselective bionanocatalysis on gold nanoparticles

As yet, different enzymes were immobilized on gold nanoparticles both through adsorption and covalent binding. However, there is only one evaluation if such immobilization influenced enzyme enantioselectivity, which is an essential parameter in biocatalysis. Therefore systematic studies with enzymes immobilized on gold nanoparticles through covalent binding and embedded through adsorption were performed. Adsorption was not efficient method and it significantly lowered enantioselectivity of enzymes. In turn, covalent binding was in most cases very good method of immobilization, especially for Pseudomonas cepacia lipase, where conversion and enantioselectivity were even slightly better than for native enzyme. It was also evaluated that in case of adsorption size of nanoparticles did not influence enantioselectivity, but in case of covalent binding small nanoparticles gave much better results than big ones.     

A ubiquitin conjugating enzyme encoded by African swine fever virus.

The post-translational modification of proteins by covalent attachment of ubiquitin occurs in all eukaryotes by a multi-step process. A family of E2 or ubiquitin conjugating (UBC) enzymes catalyse one step of this process and these have been implicated in several diverse regulatory functions. We report here the sequence of a gene encoded by African swine fever virus (ASFV) which has high homology with UBC enzymes. This ASFV encoded enzyme has UBC activity when expressed in Escherichia coli since it forms thiolester bonds with [125I]ubiquitin in the presence of purified ubiquitin activating enzyme (E1) and ATP, and subsequently transfers [125I]ubiquitin to specific protein substrates. These substrates include histones, ubiquitin and the UBC enzyme itself. The ASFV encoded UBC enzyme is similar in structure and enzyme activity to the yeast ubiquitin conjugating enzymes UBC2 and UBC3. This is the first report of a virus encoding a functionally active UBC enzyme and provides an example of the exploitation of host regulatory mechanisms by viruses.     

Experimental evidence for a zero-order ultrasensitivity in a simple substrate cycle

It was shown [Goldbeter & Koshland (1981), Proc. Natl. Acad. Sci. USA, 78, 6840-6844] that amplified sensitivity may arise in reversible covalent modification systems, when the converter enzymes operate in their zero-order region. We show that "zero-order ultrasensitivity" may also occur in simple substrate cycles. The experimental study deals with the Formate/Lactic dehydrogenases model cycle, interconverting the reduced and oxidized forms of NAD. For NAD(H) concentrations high enough (with respect to the enzyme KM's), abrupt changes in the steady-state substrate concentrations may result from small variations in the ratio of maximal enzyme activities. The amplification factors are measured. Implications in metabolic regulation are also taken up.     

Metabolomics‐driven quantitative analysis of ammonia assimilation in E. coli

Despite extensive study of individual enzymes and their organization into pathways, the means by which enzyme networks control metabolite concentrations and fluxes in cells remains incompletely understood. Here, we examine the integrated regulation of central nitrogen metabolism in Escherichia coli through metabolomics and ordinary‐differential‐equation‐based modeling. Metabolome changes triggered by modulating extracellular ammonium centered around two key intermediates in nitrogen assimilation, α‐ketoglutarate and glutamine. Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes. Combined with covalent modification reactions controlling glutamine synthetase activity, such active‐site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.     

Comparison of native and recombinant chlorite dismutase from Ideonella dechloratans.

A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme. Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis. Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein. Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein. Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low pK(a). The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination. These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions.     

S100A7, S100A10, and S100A11 are transglutaminase substrates

S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.     

Simple Biochemical Pathways far from Steady State Can Provide Switchlike and Integrated Responses

Covalent modification cycles (systems in which the activity of a substrate is regulated by the action of two opposing enzymes) and ligand/receptor interactions are ubiquitous in signaling systems and their steady-state properties are well understood. However, the behavior of such systems far from steady state remains unclear. Here, we analyze the properties of covalent modification cycles and ligand/receptor interactions driven by the accumulation of the activating enzyme and the ligand, respectively. We show that for a large range of parameters both systems produce sharp switchlike response and yet allow for temporal integration of the signal, two desirable signaling properties. Ultrasensitivity is observed also in a region of parameters where the steady-state response is hyperbolic. The temporal integration properties are tunable by regulating the levels of the deactivating enzyme and receptor, as well as by adjusting the rate of accumulation of the activating enzyme and ligand. We propose that this tunability is used to generate precise responses in signaling systems.     

Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea

Ubiquitin/ubiquitin-like (Ub/Ubl) proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1) and a JAMM/MPN+ metalloprotease (HvJAMM1). Molybdopterin (MPT) synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE) of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase) can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease.     

The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases

ATP- and NAD(+)-dependent DNA ligases, ATP-dependent RNA ligases and GTP-dependent mRNA capping enzymes comprise a superfamily of proteins that catalyze nucleotidyl transfer to polynucleotide 5' ends via covalent enzyme-(lysyl-N)-NMP intermediates. The superfamily is defined by five peptide motifs that line the nucleotide-binding pocket and contribute amino acid sidechains essential for catalysis. Early crystal structures revealed a shared core tertiary structure for DNA ligases and capping enzymes, which are composed minimally of a nucleotidyltransferase domain fused to a distal OB-fold domain. Recent structures of viral and bacterial DNA ligases, and a fungal mRNA capping enzyme underscore how the substrate-binding and chemical steps of the ligation and capping pathways are coordinated with large rearrangements of the component protein domains and with remodeling of the atomic contacts between the enzyme and the nucleotide at the active site. The first crystal structure of an RNA ligase suggests that contemporary DNA ligases, RNA ligases and RNA capping enzymes evolved by fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

Dynamin interacts with members of the sumoylation machinery

Dynamin is a GTP-binding protein whose oligomerization-dependent assembly around the necks of lipid vesicles mediates their scission from parent membranes. Dynamin is thus directly involved in the regulation of endocytosis. Sumoylation is a post-translational protein modification whereby the ubiquitin-like modifier Sumo is covalently attached to lysine residues on target proteins by a process requiring the concerted action of an activating enzyme (ubiquitin-activating enzyme), a conjugating enzyme (ubiquitin carrier protein), and a ligating enzyme (ubiquitin-protein isopeptide ligase). Here, we show that dynamin interacts with Sumo-1, Ubc9, and PIAS-1, all of which are members of the sumoylation machinery. Ubc9 and PIAS-1 are known ubiquitin carrier protein and ubiquitin-protein isopeptide ligase enzymes, respectively, for the process of sumoylation. We have identified the coiled-coil GTPase effector domain (GED) of dynamin as the site on dynamin that interacts with Sumo-1, Ubc9, and PIAS-1. Although we saw no evidence of covalent Sumo-1 attachment to dynamin, Sumo-1 and Ubc9 are shown here to inhibit the lipid-dependent oligomerization of dynamin. Expression of Sumo-1 and Ubc9 in mammalian cells down-regulated the dynamin-mediated endocytosis of transferrin, whereas dynamin-independent fluid-phase uptake was not affected. Furthermore, using high resolution NMR spectroscopy, we have identified amino acid residues on Sumo-1 that directly interact with the GED of dynamin. The results suggest that the GED of dynamin may serve as a scaffold that concentrates the sumoylation machinery in the vicinity of potential acceptor proteins.