Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.     

A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.     

Non-invasive measurement of small peptides in the common marmoset (Callithrix jacchus): a radiolabeled clearance study and endogenous excretion under varying social conditions

A non-invasive assay for measurement of oxytocin (OT) and vasopressin (AVP) in primates would enable researchers to study the relationship between the endocrine system and behavior without disturbing potentially endangered animals in their natural habitats. In order to test whether or not OT specifically would be measurable in the urine of a primate, 10 microCi of tritium-labeled OT were injected into the peripheral blood supply of four adult male common marmosets (Callithrix jacchus), with continuous urinary collection over 48 h. When urine was processed by HPLC separation and beta counting for radioactive clearance, the label was present in all samples in the fraction where OT elutes. Large amounts of OT were also seen in a fraction other than that containing the OT standard, indicating that OT is measurable but that it also undergoes substantial metabolic breakdown. In a second experiment, we isolated six common marmosets for 48 h and then exposed them to social contact to evaluate the effect of changing social stimuli on endogenous urinary measurement of both OT and AVP. Both were measured after HPLC separation to isolate the intact molecule and also to control for cross-reactivity with metabolites in subsequent RIA. Cortisol was also measured to objectively evaluate the stress response. A priori assumptions were that urinary OT and AVP would be lower during a period of isolation and higher during periods of social contact. These assumptions were met, leading us to conclude that peripheral OT and AVP are measurable via urinary assay and that such an assay is a valid means of evaluating social condition in this species.     

MORPHOLOGICAL EVOLUTION IN MUROID RODENTS I. CONSERVATIVE PATTERNS OF CRANIOMETRIC COVARIANCE AND THEIR ONTOGENETIC BASIS IN THE NEOTROPICAL GENUS ZYGODONTOMYS

Analyses of craniodental measurement data from 15 wild-collected population samples of the Neotropical muroid rodent genus Zygodontomys reveal consistent patterns of relative variability and correlation that suggest a common latent structure. Eigenanalysis of each sample covariance matrix of logarithms yields a first principal component that accounts for a large fraction of the total variance. Variances of subsequent sample principal components are much smaller, and the results of bootstrap resampling together with asymptotic statistics suggest that characteristic roots of the covariance matrix after the first are seldom distinct. The coefficients of normalized first principal components are strikingly similar from sample to sample: inner products of these vectors reveal an average between-sample correlation of 0.989, and the mean angle of divergence is only about eight degrees. Since first principal component coefficients identify the same contrasts among variables as comparisons of relative variability and correlation, we conclude that a single factor accounts for most of the common latent determination of these sample dispersions. Analyses of variance based on toothwear (a coarse index of age) and sex in the wild-collected samples, and on known age and sex in a captive-bred population, reveal that specimen scores on sample first principal components are age- and sex-dependent; residual sample dispersion, however, is essentially unaffected by age, sex, or age × sex interaction. The sample first principal component therefore reflects the covariance among measured dimensions induced by general growth, and its coefficients are interpretable as exponents of postnatal growth allometry. Path-analytic models that incorporate prior knowledge of the equivalent allometric effects of general growth within these samples can be used to decompose the between-sample variance by factors corresponding to other ontogenetic mechanisms of form change. The genetic or environmental determinants of differences in sample mean phenotypes induced by such mechanisms, however, can be demonstrated only by experiment.     

Improving Risk Assessment: Priorities for Epidemiologic Research

The Epidemiology Work Group at the Workshop on Future Research for Improving Risk Assessment Methods, Of Mice, Men, and Models, held August 16 to 18, 2000, at Snowmass Village, Aspen, Colorado, concluded that in order to improve the utility of epidemiologic studies for risk assessment, methodologic research is needed in the following areas: (1) aspects of epidemiologic study designs that affect doseresponse estimation; (2) alternative methods for estimating dose in human studies; and (3) refined methods for dose-response modeling for epidemiologic data. Needed research in aspects of epidemiologic study design includes recognition and control of study biases, identification of susceptible subpopulations, choice of exposure metrics, and choice of epidemiologic risk parameters. Much of this research can be done with existing data. Research needed to improve determinants of dose in human studies includes additional individual-level data (e.g., diet, co-morbidity), development of more extensive human data for physiologically based pharmacokinetic (PBPK) dose modeling, tissue registries to increase the availability of tissue for studies of exposure/dose and susceptibility biomarkers, and biomarker data to assess exposures in humans and animals. Research needed on dose-response modeling of human studies includes more widespread application of flexible statistical methods (e.g., general additive models), development of methods to compensate for epidemiologic bias in dose-response models, improved biological models using human data, and evaluation of the benchmark dose using human data. There was consensus among the Work Group that, whereas most prior risk assessments have focused on cancer, there is a growing need for applications to other health outcomes. Developmental and reproductive effects, injuries, respiratory disease, and cardiovascular disease were identified as especially high priorities for research. It was also a consensus view that epidemiologists, industrial hygienists, and other scientists focusing on human data need to play a stronger role throughout the risk assessment process. Finally, the group agreed that there was a need to improve risk communication, particularly on uncertainty inherent in risk assessments that use epidemiologic data.     

Reciprocal control of pyruvate dehydrogenase kinase and phosphatase by inositol phosphoglycans. Dynamic state set by "push-pull" system

Reversible phosphorylation of proteins regulates numerous aspects of cell function, and abnormal phosphorylation is causal in many diseases. Pyruvate dehydrogenase complex (PDC) is central to the regulation of glucose homeostasis. PDC exists in a dynamic equilibrium between de-phospho-(active) and phosphorylated (inactive) forms controlled by pyruvate dehydrogenase phosphatases (PDP1,2) and pyruvate dehydrogenase kinases (PDK1-4). In contrast to the reciprocal regulation of the phospho-/de-phospho cycle of PDC and at the level of expression of the isoforms of PDK and PDP regulated by hormones and diet, there is scant evidence for regulatory factors acting in vivo as reciprocal "on-off" switches. Here we show that the putative insulin mediator inositol phosphoglycan P-type (IPG-P) has a sigmoidal inhibitory action on PDK in addition to its known linear stimulation of PDP. Thus, at critical levels of IPG-P, this sigmoidal/linear model markedly enhances the switchover from the inactive to the active form of PDC, a "push-pull" system that, combined with the developmental and hormonal control of IPG-P, indicates their powerful regulatory function. The release of IPGs from cell membranes by insulin is significant in relation to diabetes. The chelation of IPGs with Mn2+ and Zn2+ suggests a role as "catalytic chelators" coordinating the traffic of metal ions in cells. Synthetic inositol hexosamine analogues are shown here to have a similar linear/sigmoidal reciprocal action on PDC exerting push-pull effects, suggesting their potential for treatment of metabolic disorders, including diabetes.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.     

Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215–217 β-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* → E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.Thrombin is the pivotal protease of blood coagulation and is endowed with both procoagulant and anticoagulant roles in vivo (1). Thrombin acts as a procoagulant when it converts fibrinogen into an insoluble fibrin clot, activates clotting factors V, VIII, XI, and XIII, and cleaves PAR1² and PAR4 on the surface of human platelets thereby promoting platelet aggregation (2). Upon binding to thrombomodulin, a receptor present on the membrane of endothelial cells, thrombin becomes unable to interact with fibrinogen and PAR1 but increases >1,000-fold its activity toward the zymogen protein C (3). Activated protein C generated from the thrombin-thrombomodulin complex down-regulates both the amplification and progression of the coagulation cascade (3) and acts as a potent cytoprotective agent upon engagement of EPCR and PAR1 (4).The dual nature of thrombin has long motivated interest in dissociating its procoagulant and anticoagulant activities (5–12). Thrombin mutants with anticoagulant activity help rationalize the bleeding phenotypes of several naturally occurring mutations and could eventually provide new tools for pharmacological intervention (13) by exploiting the natural protein C pathway (3, 14, 15). Previous mutagenesis studies have led to the identification of the E217A and E217K mutations that significantly shift thrombin specificity from fibrinogen toward protein C relative to the wild type (10–12). Both constructs were found to display anticoagulant activity in vivo (10, 12). The subsequent discovery of the role of Trp-215 in controlling the balance between pro- and anti-coagulant activities of thrombin (16) made it possible to construct the double mutant W215A/E217A (WE) featuring >19,000-fold reduced activity toward fibrinogen but only 7-fold loss of activity toward protein C (7). These properties make WE the most potent anticoagulant thrombin mutant engineered to date and a prototype for a new class of anticoagulants (13). In vivo studies have revealed an extraordinary potency, efficacy, and safety profile of WE when compared with direct administration of activated protein C or heparin (17–19). Importantly, WE elicits cytoprotective effects (20) and acts as an antithrombotic by antagonizing the platelet receptor GpIb in its interaction with von Willebrand factor (21).What is the molecular mechanism underscoring the remarkable functional properties of WE? The mutant features very low activity toward synthetic and physiological substrates, including protein C. However, in the presence of thrombomodulin, protein C is activated efficiently (7). A possible explanation is that WE assumes an inactive conformation when free but is converted into an active form in the presence of thrombomodulin. The ability of WE to switch from inactive to active forms is consistent with recent kinetic (22) and structural (23, 24) evidence of the significant plasticity of the trypsin fold. The active form of the protease, E, coexists with an inactive form, E*, that is distinct from the zymogen conformation (25). Biological activity of the protease depends on the equilibrium distribution of E* and E, which is obviously different for different proteases depending on their physiological role and environmental conditions (25). The E* form features a collapse of the 215–217 β-strand into the active site and a flip of the peptide bond between residues Glu-192 and Gly-193, which disrupts the oxyanion hole. These changes have been documented crystallographically in thrombin and other trypsin-like proteases such as αI-tryptase (26), the high temperature requirement-like protease (27), complement factor D (28), granzyme K (29), hepatocyte growth factor activator (30), prostate kallikrein (31), prostasin (32, 33), complement factor B (34), and the arterivirus protease nsp4 (35). Hence, the questions that arise about the molecular mechanism of WE function are whether the mutant is indeed stabilized in the inactive E* form and whether it can be converted to the active E form upon thrombomodulin binding.Structural studies of the anticoagulant mutants E217K (36) and WE (37) show a partial collapse of the 215–217 β-strand into the active site that abrogates substrate binding. The collapse is similar to, but less pronounced than, that observed in the structure of the inactive E* form of thrombin where Trp-215 relinquishes its hydrophobic interaction with Phe-227 to engage the catalytic His-57 and residues of the 60-loop after a 10 Å shift in its position (24). These more substantial changes have been observed recently in the structure of the anticoagulant mutant Δ146–149e (38), which has proved that stabilization of E* is indeed a molecular mechanism capable of switching thrombin into an anticoagulant. It would be simple to assume that both E217K and WE, like Δ146–149e, are stabilized in the E* form. However, unlike Δ146–149e, both E217K and WE carry substitutions in the critical 215–217 β-strand that could result into additional functional effects overlapping with or mimicking a perturbation of the E*-E equilibrium. A significant concern is that both structures suffer from crystal packing interactions that may have biased the conformation of side chains and loops near the active site (24). The collapsed structures of E217K and WE may be artifactual unless validated by additional structural studies where crystal packing is substantially different.To address the second question, kinetic measurements of chromogenic substrate hydrolysis by WE in the presence of saturating amounts of thrombomodulin have been carried out (37), but these show only a modest improvement of the k_cat/K_m as opposed to >57,000-fold increase observed when protein C is used as a substrate (7, 37). The modest effect of thrombomodulin on the hydrolysis of chromogenic substrates is practically identical to that seen upon binding of hirugen to exosite I (37) and echoes the results obtained with the wild type (39) and other anticoagulant thrombin mutants (7, 9, 10, 12, 38). That argues against the ability of thrombomodulin alone to significantly shift the E*-E equilibrium in favor of the E form. Binding of a fragment of the platelet receptor PAR1 to exosite I in the D102N mutant stabilized in the E* form (24) does trigger the transition to the E form (23), but evidence that a similar long-range effect exists for the E217K or WE mutants has not been presented.In this study we have addressed the two unresolved questions about the mechanism of action of the anticoagulant thrombin mutant WE. Here we present new structures of the mutant in its human and murine versions, free and bound to a fragment of the thrombin receptor PAR1 at exosite I. The structures are complemented by direct energetic assessment of the binding of ligands to exosite I and its effect on the E*-E equilibrium.