SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.     

Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data

OBJECTIVE: We used a new graphical ordinal logit method (GOLDminer) to assess a single cardiac troponin T (cTnT) analysis at the time of admission (first generation monoclonal; Roche BMC Corp., Indianapolis, Indiana), the character of chest pain, and electrocardiographic (ECG)findings in predicting the likelihood of acute myocardial infarction (AMI) in patients presenting with suspected myocardial ischemia. The final diagnosis of AMI was based on serial ECG findings and evolution of CKMB isoenzyme levels in conjunction with clinical findings. SUBJECTS: The study population consisted of 293 consecutive patients who presented at a mean of six hours after onset of chest pain or associated symptoms warranting a "rule-out" for AMI assessment to a university-affiliated community hospital. RESULTS: The odds-ratio for an elevated cTnT (> 0. 1 ng/ml) in AMI was 22.2:1. There was an association between typical chest pain and cTnT (chi square = 78.23, p < .0001) and between abnormal ECG findings and cTnT (chi square = 108, p < .0001). The cTnT yielded diagnostic benefit in addition to chest pain characteristics and ECG findings in AMI. We present the odds-ratios for the combined features in GOLDminer plots. CONCLUSION: We demonstrate how the odds-ratios for AMI are obtained after scaling continuous to ordinal the values for a single cTnT determination alone and with other features in patients presenting with chest pain.     

Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory

OBJECTIVE: We examine the use of information theory applied to a single cardiac troponin T (cTnT) (first generation monoclonal; Boehringer Mannheim Corp., Indianapolis, Indiana) used with the character of chest pain, electrocardiography (ECG) and serial ECG changes in the evaluation of acute myocardial infarction (AMI). We combined a single measure of cTnT (blinded to the investigators) with a creatine kinase MB isoenzyme (CK-MB) measurement to discover the best decision value for this test in a study of 293 consecutive patients presenting to the emergency department with symptoms warranting exclusion of AMI. METHODS: The decision value for determining whether cTnT is positive or negative was determined independently of the final diagnosis by examining the information in the cTnT and CKMB data. Using information theory, an autocorrelation matrix with a one-to-one pairing of the CKMB and troponin T was constructed. The effective information, also known as Kullback entropy, assigned the values for troponin T and for CKMB that have the lowest frequency of misclassification error. The Kullback entropy is determined by subtracting the data entropy from the maximum entropy of the data set in which the information has been destroyed. The assignment of the optimum decision values was made independently of the clinical diagnoses without the construction of a receiver-operator characteristic curve (ROC). The final diagnosis of AMI was independently determined by the clinicians and entered into the medical record. RESULTS: The decision value for cTnT was 0.1 ng/ml as determined by the the information in the data. The method was validated within the same study by mapping the results so obtained into the diagnoses obtained independently by the clinicians using all of the methods at their disposal. The cTnT was different in AMI (n = 60) compared with non-AMI patients (n = 233) (2.08 +/- 0.21 vs. 0.07 +/- 0.10; p < .0001). CONCLUSION: Information theory provides a strong framework and methodology for determining the decision value for cTnT which minimizes misclassification errors at 0.1 ng/ml. The result has a strong correlation with other features in detecting AMI in patients presenting with chest pain.     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.     

Protease inhibitors and directed evolution: enhancing plant resistance to nematodes

Plant nematodes are agricultural pests, the control of which relies on chemical nematicides and fumigants that are among the most toxic and environmentally damaging of all agrochemicals. New approaches to control, based on transgenic resistance, would provide important health and environmental benefits. In this chapter we consider briefly some targets for engineering nematode resistance and discuss the use of plant protease inhibitors as anti-feedants. This approach has provided plants that display good levels of resistance against a range of nematode species. To enhance this defence strategy further we are investigating the value of directed evolution to improve the characteristics of protease inhibitors. We describe the approaches of DNA shuffling and phage display that are being used to create and screen variant libraries in the search for inhibitors with improved features.     

Role of the proline-rich domain of dynamin-2 and its interactions with Src homology 3 domains during endocytosis of the AT1 angiotensin receptor

In nonneural tissues, the dynamin-2 isoform participates in the formation of clathrin-coated vesicles during receptor endocytosis. In this study, the mechanism of dynamin-2 action was explored during endocytosis of the G protein-coupled AT1A angiotensin receptor expressed in Chinese hamster ovary cells. Dynamin-2 molecules with mutant pleckstrin homology domains or deleted proline-rich domains (PRD) exerted dominant negative inhibition on the endocytosis of radiolabeled angiotensin II. However, only the PRD mutation interfered with the localization of the dynamin-2 molecule to clathrin-coated pits and reduced the inhibitory effect of the GTPase-deficient K44A mutant dynamin-2. Green fluorescent protein-tagged Src homology 3 (SH3) domains of endophilin I and amphiphysin II, two major binding partners of dynamins, also inhibited AT1A receptor-mediated endocytosis of angiotensin II. These effects were partially or fully, respectively, restored by the overexpression of dynamin-2. Transient overexpression of these SH3 domains also reduced the localization of dynamin-2 to clathrin-coated pits. These data indicate that, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles, interaction of the dynamin-2 PRD with SH3 domains of proteins such as the amphiphysins and endophilins is essential for AT1A receptor endocytosis. This mechanism could be of general importance in dynamin-dependent endocytosis of other G protein-coupled receptors in nonneural tissues.     

The crystallographic structure of brome mosaic virus

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.