Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.     

The evolutionary consequences of plasticity in host-pathogen interactions

Interactions between individuals such as hosts and pathogens are often characterized by substantial phenotypic plasticity. Pathogens sometimes alter their exploitation strategies in response to defensive strategies adopted by their host and vice versa. Nevertheless, most game-theoretic models developed to explain the evolution of pathogen and host characteristics assume that no such plasticity occurs. Allowing for phenotypic plasticity in these models is difficult because one must focus on the evolution of pathogen and host reaction norms, and then allow for the potentially indefinite reciprocal changes in pathogen and host behaviour that occur during an infection as a result of their interacting reaction norms. Here, we begin to address these issues for a simple host-pathogen system in which the pathogen exhibits a level of virulence and the host exhibits a level of immune clearance. We find, quite generally, that plasticity promotes the evolution of higher levels of cooperation, in this case leading to reduced levels of both virulence and clearance.     

The function of conserved cysteine residues in the extracellular domain of human receptor-activity-modifying protein

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

毕赤酵母高效表达策略概述

毕赤酵母表达系统是外源蛋白表达的较为理想的系统,但是并不是所有蛋白都能利用此系统获得高效表达,不同来源的蛋白,其表达水平、生物活性和稳定性均存有明显差别。概述了影响毕赤酵母高效表达的主要因素以及外源蛋白在毕赤酵母中的高效表达策略。     

Structural features of plant chitinases and chitin-binding proteins

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three-dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively.     

Genetic and evolutionary consequences of symbiosis

Permanent stable symbioses, primarily microbial, are analyzed as parasexual phenomena from the evolutionary point of view. Such associations bring together in single individuals heritable traits of high selective advantage in certain environments. By convergent evolution several types of associations have repeatedly arisen: motile photosynthetic forms, nitrogen fixing and wood digesting complexes, and so forth. Many examples are discussed from the point of view of the number of originally independent genomes that comprise the recognizable individuals.The level of partner integration in many associations is analyzed. Examples of many levels: genic, gene product, metabolite, behavioral, and the methods by which they can be distinguished are discussed.The literature concerning a large number of associations is reviewed: Methanobacillus; predatory and consortia bacteria; blue green algal sheath dwelling bacteria; anaerobic worm-bacterial; algal, and foreign chloroplast retention by heterotrophic eukaryotes (ciliates, coelenterates, mollusks); the double nucleated photosynthetic dinoflagellate (Peridinium balticum); hindgut microbes in termites and woodroaches (Pyrsonympha, Barbulanympha and their associated spirochetes and other bacteria); sand dwelling and other ciliates and their associated bacteria; and so forth. The status of observations and artificial systems claiming evidence for transfer of genes between very distantly related organisms is critically discussed.A continuum from nearly completely autonomous partners (e.g., zoochlorellae in invertebrate animals) to nearly unrecognizable merged components (e.g., gamma particles in Blastocladiella) is found to exist among examples of extant organisms. The diversity and prevalence of such associations support the concept that there are many precedents for the steps hypothesized in the serial endosymbiotic theory of the origin of eukaryotic cells.     

Human evolutionary research in eastern germany

Human evolutionary research in Eastern Germany, limited in this paper to the origins and evolution of Pleistocene man, has mainly focused during the last decade on theoretical aspects of the evolution of man and human society from multiple and interdisciplinary perspectives. An interdisciplinary working group ‘Probleme der Menschwerdung’ was founded in 1977. About 30 specialists from various fields have discussed problems of the origins and evolution of man and human society under interdisciplinary aspects. The activities, difficulties and results of this cooperation will be outlined in this paper. There will also be given a progress report on the disciplinary initiated research on local sites as well as on general aspects and problems. Paper dedicated to Professor Dr. rer. nat. Dr.sc.med. Hans Grimm, Berlin, on his 80th birthday, February 7, 1990.     

Structural and energetic consequences of mutations in a solvated hydrophobic cavity

The structural and energetic consequences of modifications to the hydrophobic cavity of interleukin 1-beta (IL-1beta) are described. Previous reports demonstrated that the entirely hydrophobic cavity of IL-1beta contains positionally disordered water. To gain a better understanding of the nature of this cavity and the water therein, a number of mutant proteins were constructed by site-directed mutagenesis, designed to result in altered hydrophobicity of the cavity. These mutations involve the replacement of specific phenylalanine residues, which circumscribe the cavity, with tyrosine, tryptophan, leucine and isoleucine. Using differential scanning calorimetry to determine the relative stabilities of the wild-type and mutant proteins, we found all of the mutants to be destabilizing. X-ray crystallography was used to identify the structural consequences of the mutations. No clear correlation between the hydrophobicities of the specific side-chains introduced and the resulting stabilities was found.     

Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors

Relatively little information is available about the molecular mechanism of ethanol inhibition of P2X receptors. Here, we investigated the possibility that 10 conserved cysteine residues in the extracellular loop of the rat P2X₄ receptor may regulate ethanol inhibition of the receptor using a series of individual cysteine to alanine point mutations. Each of the mutated receptors generated robust inward current in response to ATP and the mutations produced less than a sixfold change in the ATP EC₅₀ value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current activated by an EC₄₀ concentration of ATP. By contrast, for the C261A and C270A mutants, ethanol inhibited ATP-activated current in a competitive manner similar to that for the wild-type receptor. Interestingly, for the C132A, C159A, C217A, and C227A mutants, ethanol inhibited ATP-activated current, but decreased the maximal response to ATP by 70-75% without significantly changing the EC₅₀ value of ATP, thus exhibiting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X₄ receptor by ethanol.     

Identification of potential redox-sensitive cysteines in cytosolic forms of fructosebisphosphatase and glyceraldehyde-3-phosphate dehydrogenase

Tertiary-structure modeling suggests the occurrence of disulfide bonds in the cytosolic form of fructosebisphosphatase (EC 3.1.3.11) in spinach (Spinacia oleracea L.), sugarbeet (Beta vulgaris L.) and potato (Solanum tuberosum L.). Redox modulation could then control the AMP sensitivity of fructosebisphosphatase in the cytosol, as suggested by the experiments of E. Khayat et al. (1993, Plant Physiol. 101, 57–64). Modeling also reveals two cysteine residues correctly positioned to form a disulfide bond and hence potentially redox-sensitive in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from the facultative crassulacean metabolism plant Mesembryanthemum crystallinum L.Abbreviations Cys cysteine - DTT dithiothreitolThis work was supported by NSF DCB-9018265 and US Department of Energy, Office of Health and Environmental Research, under contract W-31-109-ENG-38, and by laboratory-directed research and development funding from Argonne National Laboratory. We thank Hans J. Bohnert, University of Arizona, for providing the ice plant seeds, Xiaomu Niu and Paul M. Hasegawa, Purdue University, for providing the salt bush roots, Larry Sykora and staff at the UIC Greenhouse for cultivating the ice plants, and Christie Aljets for assistance with some of the activity determinations.     

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.     

On the cysteine and cystine content of proteins

Summary Analysis of published data on the cysteine and half-cystine content of proteins indicates that most intracellular proteins may be classified as sulfhydryl proteins (those containing cysteine but little or no half-cystine) and that such sulf-hydryl proteins have a low cysteine content. The mean cysteine content found for 32 intracellular mammalian proteins was 1.6 % and intracellular proteins of many bacteria have similar or lower values. Extracellular mammalian proteins are primarily disulfide proteins (those containing half-cystine but little or no cysteine) and have a high half-cystine content, the mean value found for some 34 extracellular mammalian proteins being 4.1 %. This is contrasted with many of the extracellular proteins from facultative bacteria which are cyst(e)ine-free proteins, being lacking in both cysteine and half-cystine. These and related observations are interpreted in terms of the evolution of life in a reducing atmosphere and the subsequent transition to an oxidizing environment. It is suggested that disulfide proteins evolved primarily after the accumulation of oxygen in the atmosphere.     

Over expression and purification of recombinant human myocilin

Human myocilin is a 55 kDa protein that is implicated in primary open angle glaucoma (POAG). Understanding the structure and folding of the native protein and the mutants that increase aggregation could lead to possible prevention of the condition. We report here the over expression and purification of the human myocilin in E. coli. The initial expression of recombinant myocilin in E. coli was found to be low. The problem of low yield was found to be due to multiple causes and was overcome using a suitable combination of vectors, tags, host background and expression protocols. The overexpressed human myocilin was purified by affinity column chromatography to yield about 8 mg of protein from 1 l of culture. The protein purity and folding were confirmed using electrophoresis, immunoblotting and fluorescence spectroscopy. Further biophysical characterization and crystallization trials using the recombinant human myocilin will pave the way for better understanding of the structure–aggregation relationship that is involved in causing POAG.     

Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1beta by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1beta production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode.     

The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser_108/121, HB-EGF-Cys/Ser_116/132, and HB-EGF-Cys/Ser_134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M_r of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser_108/121 and HB-EGF-Cys/Ser_116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF_134/143 proteins competed with ¹²⁵I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser_108/121 and HB-EGF-Cys/Ser_116/132 failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser_134/143 antagonizes EGFRs.     

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.