Mapping out starvation responses

What are the pathways that underlie the coordinated responses of an organism to well-fed and food-deprived states? A report in this issue of Cell Metabolism suggests that starvation functions via a muscarinic acetylcholine receptor to activate MAP kinase signaling in the pharyngeal muscle of C. elegans (You et al., 2006).     

Quartz crystal microbalance studies of multilayer glucagon fibrillation at the solid-liquid interface

We have used a quartz crystal microbalance with dissipation (QCM-D) to monitor the changes in layer thickness and viscoelastic properties accompanying multilayer amyloid deposition in situ for the first time. By means of atomic force microscope imaging, an unequivocal correlation is established between the interfacial nucleation and growth of glucagon fibrils and the QCM-D response. The combination of the two techniques allows us to study the temporal evolution of the interfacial fibrillation process. We have modeled the QCM-D data using an extension to the Kelvin-Voigt viscoelastic model. Three phases were observed in the fibrillation process: 1), a rigid multilayer of glucagon monomers forms and slowly rearranges; 2), this multilayer subsequently evolves into a dramatically more viscoelastic layer, containing a polymorphic network of micrometer-long fibrils growing from multiple nucleation sites; and 3), the fibrillar formation effectively stops as a result of the depletion of bulk-phase monomers, although the process can be continued without a lag phase by subsequent addition of fresh monomers. The robustness of the QCM-D technique, consolidated by complementary atomic force microscope studies, should make it possible to combine different components thought to be involved in the plaque formation process and thus build up realistic models of amyloid plaque formation in vitro.     

Spectroscopic evidence for the existence of an obligate pre-fibrillar oligomer during glucagon fibrillation

The 29-residue peptide hormone glucagon has been used as a model system for the study of amyloid-like fibrils. Atomic force microscopy (AFM) studies have detected putative oligomeric species during this lag phase, but this has not been confirmed by any spectroscopic technique. Here we use an attached pyrene group to detect association (excimer formation) between individual glucagon molecules. Our data show that excimer formation precedes fibrillation both at different pHs and with sulfate, and support our original proposal that glucagon fibril formation is preceded by oligomer formation. We suggest that pyrene-labelling may be a useful way to monitor oligomer formation during protein fibrillation.     

N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.     

The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.     

Mapping out the roles of MAP kinases in plant defense.

Arabidopsis contains 20 MAP kinase genes, but their roles in plant physiology have remained largely unknown because of a lack of mutants. Recent papers from two groups have shed new light on the function of two different MAP kinases. The Arabidopsis MPK4 gene appears to negatively regulate salicylic acid-mediated defense responses and positively regulate jasmonic acid-induced responses. The tobacco SIPK gene (orthologous to Arabidopsis MPK6) appears to positively regulate programmed cell death.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.     

Photoaffinity labeling of the glucagon receptor with a new glucagon analog

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.     

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs

Summary Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon_1–21 and glucagon_1–6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon_2–29 ([1-des-histidine]-glucagon), [1-N-carbamoylhistidine]-glucagon and [1-N-carbamoylhistidine, 12-N-carbamoyllysine]-glucagon are all partial agonists.These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-N-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA₂ values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals.Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.     

Low-dose-rate extrapolation using the multistage model

The distribution of the maximum likelihood estimates of virtually safe levels of exposure to environmental chemicals is derived by using large-sample theory and Monte Carlo simulation according to the Armitage-Doll multistage model. Using historical dose-response we develop a set of 33 two-stage models upon which we base our conclusions. The large-sample distributions of the virtually safe dose are normal for cases in which the multistage-model parameters have nonzero expectation, and are skewed in other cases. The large-sample theory does not provide a good approximation of the distribution observed for small bioassays when Monte Carlo simulation is used. The constrained nature of the multistage-model parameters leads to bimodal distributions for small bioassays. The two modes are the direct result of estimating the linear parameter in the multistage model; the lower mode results from estimating this parameter to be nonzero, and the upper mode from estimating it to be zero. The results of this research emphasize the need for incorporation of the biological theory in the model-selection process.     

冷冻电镜单颗粒三维重构密度掩模的自动生成方法研究

冷冻电镜单颗粒三维重构技术是用来解析生物大分子三维结构的常用方法.然而目前在单颗粒三维重构过程中,溶剂平滑操作还存在一定缺陷:没有一款主流的单颗粒三维重构程序能够自动寻找掩模(mask)三维密度图,使得三维重构过程难免受到噪音统计学模型计算偏差的干扰.为解决这一问题,本研究借鉴X射线晶体学中解析优化相位所广泛采用的溶剂平滑方法,采用高斯滤波、坎尼边缘检测、最小误差阈值处理等方法处理重构所得三维密度图,优化溶剂平滑操作,发展在单颗粒三维重构过程中自动寻找mask三维密度图的方法.运用三维密度图傅里叶壳层相关系数(fourier shell correlation,FSC)曲线图、模拟颗粒数据重构角度误差散点图等指标评估此方法的效果.结果表明,自动寻找mask密度图的方法能够较好地找到涵盖分子结构信号区域的mask密度图,较为明显提高三维重构所得密度图分辨率.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

Interactions of glucagon and glucagon analogs with isolated canine hepatocytes

We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.