A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88–100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.

A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway

Continuous growth and development in plants are accomplished by meristems, groups of undifferentiated cells that persist as stem cells and initiate organs. While the structures of the apical and floral meristems in dicotyledonous plants have been well described, little is known about the underlying molecular mechanisms controlling cell proliferation and differentiation in these structures. We have shown previously that the CLAVATA1 (CLV1) gene in Arabidopsis encodes a receptor kinase-like protein that controls the size of the apical and floral meristems. Here, we show that KAPP, a gene encoding a kinase-associated protein phosphatase, is expressed in apical and young floral meristems, along with CLV1. Overexpression of KAPP mimics the clv1 mutant phenotype. Furthermore, CLV1 has kinase activity: it phosphorylates both itself and KAPP. Finally, KAPP binds and dephosphorylates CLV1. We present a model where KAPP functions as a negative regulator of the CLAVATA1 signal transduction pathway.

A model of excitation and adaptation in bacterial chemotaxis

Bacterial chemotaxis is widely studied because of its accessibility and because it incorporates processes that are important in a number of sensory systems: signal transduction, excitation, adaptation, and a change in behavior, all in response to stimuli. Quantitative data on the change in behavior are available for this system, and the major biochemical steps in the signal transduction/processing pathway have been identified. We have incorporated recent biochemical data into a mathematical model that can reproduce many of the major features of the intracellular response, including the change in the level of chemotactic proteins to step and ramp stimuli such as those used in experimental protocols. The interaction of the chemotactic proteins with the motor is not modeled, but we can estimate the degree of cooperativity needed to produce the observed gain under the assumption that the chemotactic proteins interact directly with the motor proteins.

From residue matching patterns to protein folding topographies: General model and bovine pancreatic trypsin inhibitor

A coarse-grained model for protein-folding dynamics is introduced based on a discretized representation of torsional modes. The model, based on the Ramachandran map of the local torsional potential surface and the class (hydrophobic/polar/neutral) of each residue, recognizes patterns of both torsional conformations and hydrophobic-polar contacts, with tolerance for imperfect patterns. It incorporates empirical rates for formation of secondary and tertiary structure. The method yields a topological representation of the evolving local torsional configuration of the folding protein, modulo the basins of the Ramachandran map. The folding process is modeled as a sequence of transitions from one contact pattern to another, as the torsional patterns evolve. We test the model by applying it to the folding process of bovine pancreatic trypsin inhibitor, obtaining a kinetic description of the transitions between the contact patterns visited by the protein along the dominant folding pathway. The kinetics and detailed balance make it possible to invert the result to obtain a coarse topographic description of the potential energy surface along the dominant folding pathway, in effect to go backward or forward between a topological representation of the chain conformation and a topographical description of the potential energy surface governing the folding process. As a result, the strong structure-seeking character of bovine pancreatic trypsin inhibitor and the principal features of its folding pathway are reproduced in a reasonably quantitative way.

The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato

The Pto gene encodes a serine/threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto. Partial characterization of the Pto signal transduction pathway and the availability of transgenic tomato lines (± Pto) make this an ideal system for exploring the molecular basis of disease resistance. In this paper, we test two transgenic tomato cell suspension cultures (±Pto) for production of H2O2 following independent challenge with two strains of P. syringae pv. tomato (±avrPto). Only when Pto and avrPto are present in the corresponding organisms are two distinct phases of the oxidative burst seen, a rapid first burst followed by a slower and more prolonged second burst. In the remaining three plant–pathogen interactions, we observe either no burst or only a first burst, indicating that the second burst is correlated with disease resistance. Further support for this observation comes from the finding that both resistant and susceptible tomato lines produce the critical second oxidative burst when challenged with P. syringae pv. tabaci, a nonhost pathogen that elicits a hypersensitive response on both tomato lines. The Pto kinase is not required, however, for the oxidative burst initiated by non-specific elicitors such as oligogalacturonides or osmotic stress. A model describing a possible role for the Pto kinase in the overall scheme of oxidative burst signaling is proposed.

Prolonged activation of the N-methyl-d-aspartate receptor–Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current–clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-d-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA–Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.

Eps15 Is Recruited to the Plasma Membrane upon Epidermal Growth Factor Receptor Activation and Localizes to Components of the Endocytic Pathway during Receptor Internalization

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

How does a β-hairpin fold/unfold? Competition between topology and heterogeneity in a solvable model

We study the competition between topological effects and sequence inhomogeneities in determining the thermodynamics and the un/folding kinetics of a β-hairpin. Our work utilizes a new exactly solvable model that allows for arbitrary configurations of native contacts. In general, the competition between heterogeneity and topology results in a crossover of the dominant transition state. Interestingly, near this crossover, the single reaction coordinate picture can be seriously misleading. Our results also suggest that inferring the folding pathway from unfolding simulations is not always justified.

A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

Translocation pathway of protein substrates in ClpAP protease

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPγS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through ≈150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to β-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.