Detecting oxidative post-translational modifications in proteins

Summary. Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

Travelling of proteins through membranes: translocation into chloroplasts

 Most proteins involved in plastid biogenesis are encoded by the nuclear genome. They are synthesised in the cytosol and have to be transported toward and subsequently translocated into the organelle. This targeting and import process is initiated by a specific chloroplast-targeting signal. The targeting signal of the preprotein is recognised and modified by cytosolic proteins which function in transport toward the chloroplast and in maintaining the import-competent state of the preprotein. The precursor is transferred onto a multi-component complex in the outer envelope of the chloroplasts, which is formed by receptor proteins and the translocation channel. Some proteins, not containing transit sequences, are directly sorted into the outer membrane whereas the majority, containing transit sequences, will be translocated into the stroma. This involves the joint action of a protein complex in the outer envelope, one complex in the inner envelope, and soluble proteins in the intermembrane space and the stroma. The origin of this translocation complex following the endosymbiotic events is an unsolved question. Recent identification of homologous proteins to some members of this machinery in the cyanobacterium Synechocystis PCC6803 gives an initial insight into the origin of the translocation complex. Received: 27 December 1999 / Accepted: 29 March 2000     

Structural properties of proteins specific to the myelin sheath

Summary. The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small number of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a number of neurological diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a number of myelin-specific proteins are described. Author’s address: Dr. Petri Kursula, Department of Biochemistry, University of Oulu, FIN-90014 Oulu, Finland     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella

 Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892–20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation. Received: 6 August 1999 / Accepted: 23 November 1999     

Abnormal changes of plasma acute phase proteins in schizophrenia and the relation between schizophrenia and haptoglobin (Hp) gene

Summary. In this study we focused on detecting schizophrenia related changes of plasma proteins using proteomic technology and examining the relation between schizophrenia and haptoglobin (Hp) genotype. We investigated plasma proteins from schizophrenic subjects (n = 42) and healthy controls (n = 46) by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry. To further reveal the genetic relationship between acute phase proteins (APPs) and schizophrenia disease, we tested Hp α1/Hp α2 (Hp 1/2) polymorphism and two single nucleotide polymorphisms (SNPs) of Hp, rs2070937 and rs5473, for associations with schizophrenia in the Chinese Han population. With the relatively high number of samples for 2-DE work, we found that four proteins in the family of positive APPs were all up-regulated in patients. In genetic association study, we found significant associations existing between schizophrenia and Hp polymorphisms, Hp 1/2 and rs2070937 variants. Schizophrenia is accompanied by both an altered expression of Hp protein and a different genotype distribution of Hp gene, demonstrating that Hp is associated with schizophrenia. The results from proteomic and genomic aspects both indicate that acute phase reaction is likely to be an aetiological agent in the pathophysiology of schizophrenia, but not just an accompanying symptom. The positive APPs are schizophrenic related proteins, with the highly concordant results on four positive APPs. The first two authors contributed equally.     

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

Summary. The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.     

Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis lanata Ehrh.

 Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift. Received: 29 May 1999 / Accepted: 28 August 1999     

SVCT1 and SVCT2: key proteins for vitamin C uptake

Summary. Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin. SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed. This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates. Authors’ address: Dr. Isabella Savini, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy     

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy

Summary. Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin α-1 chain, β-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-α, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, α-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE. J. W. Yang and T. Czech have equally contributed to the paper.     

Mass-spectrometrical analysis of proteins encoded on chromosome 21 in human fetal brain

Summary. Overexpression of chromosome 21 genes is directly or indirectly responsible for the Down syndrome phenotype. In order to analyse chromosome 21 gene products (Chr21Ps), we extracted proteins from fetal human brain cortex and applied an ultracentrifugal and chromatographic prefractionation principle followed by two-dimensional gel electrophoresis (2-DE) and mass-spectrometrical analysis using high-throughput automated MALDI-TOF/TOF. Nine Chr21Ps were identified: pyridoxal kinase; superoxide dismutase [Cu/Zn] 1; carbonyl reductase 1; ES1 protein homolog, mitochondrial [Precursor]; cystathionine-beta-synthetase; T-complex protein 1, theta subunit; cystatin B; 6-phosphofructokinase; glycinamide ribonucleotide synthetase. Mass-spectrometric characterisation of Chr21Ps following separation in 2-DE gels is a useful tool for the analysis of these structures in brain, independent of antibody availability and specificity.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

The neuronal differentiation process involves a series of antioxidant proteins

Summary. Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.     

The relationship between albumin, other plasma proteins and variables, and age in the acute phase response after liver resection in man

Summary. A large series of plasma albumin (ALB, g/dl) and simultaneous blood and clinical measurements were prospectively performed on 92 liver resection patients, and processed to assess the correlations between ALB, other plasma proteins, additional variables and clinical events. The measurements were performed preoperatively and at postoperative day 1, 3 and 7 in all patients, and subsequently only in those who developed complications or died. In patients who recovered normally ALB was 4.3 ± 0.4 g/dl (mean ± SD) preoperatively, 3.7 ± 0.7 at day 1 and 3, and 3.9 ± 0.4 at day 7. In patients with complications its decrease was more prolonged. In non-survivors it was 3.4 ± 0.4 preoperatively, 3.0 ± 0.4 at day 1, and then decreased further. Regression analysis showed direct correlations between ALB and pseudo-cholinesterase (CHE, U/l, nv 5300-13000), cholesterol (CHOL, mg/dl), iron binding capacity (IBC, mg/dl), prothrombin activity (PA, % of standard reference) and fibrinogen, an inverse correlation with blood urea nitrogen (BUN, mg/dl) for any given creatinine level (CREAT, mg/dl), and weaker direct correlations with hematocrit, other variables and dose of exogenous albumin. An inverse relationship found between ALB and age (AGE, years) became postoperatively (POSTOP) also a function of outcome, showing larger age-related decreases in ALB associated with complications (COMPL: sepsis, liver insufficiency) or death (DEATH). Main overall correlations: CHE = 287.4(2.014)^ALB, r = 0.73; CHOL = 16.5(1.610)^ALB (1.001)^ALKPH, r = 0.71; IBC = 68.6(1.391)^ALB, r = 0.64; PA = 13.8 + 16.0(ALB), r = 0.51; BUN = 21.3 + 20.2(CREAT) – 6.2(ALB), r = 0.91; ALB = 5.0–0.013(AGE) – {0.5 + 0.003(AGE) _COMPL + 0.012(AGE) _DEATH } _POSTOP , r = 0.74 [p < 0.001 for each regression and each coefficient; ALKPH = alkaline phosphatase, U/l, nv 98-279, independent determinant of CHOL; discontinuous variables in italics label the change in regression slope or intercept associated with the corresponding condition]. These results suggest that altered albumin synthesis (or altered synthesis unable to compensate for albumin loss, catabolism or redistribution) is an important determinant of hypoalbuminemia after hepatectomy. The correlations with age and postoperative outcome support the concept that hypoalbuminemia is a marker of pathophysiologic frailty associated with increasing age, and amplified by the challenges of postoperative illness.     

14-3-3 proteins are involved in the regulation of mammalian cell proliferation

Summary. The 14-3-3 proteins are a family of abundant, widely expressed acidic polypeptides. The seven isoforms interact with over 70 different proteins. 14-3-3 isoforms have been demonstrated to be involved in the control of positive as well as negative regulators of mammalian cell proliferation. Here we used the approach of inactivating 14-3-3 protein functions via overexpression of dominant negative mutants to analyse the role of 14-3-3 proteins in mammalian cell proliferation. We found 14-3-3 dominant negative mutants to downregulate the proliferation rates of HeLa cells. Overexpression of these dominant negative mutants triggers upregulation of the protein levels of the cyclin-dependent kinase inhibitor p27, a major negative cell cycle regulator. In addition, they downregulate the protein levels of the important cell cycle promoter cyclin D1. These data provide new insights into mammalian cell proliferation control and allow a better understanding of the functions of 14-3-3 proteins.     

粉尘螨变应原第7组分全长基因序列测定与分析

目的：获得粉尘螨变应原第7组分编码基因并了解其分子特征。方法：根据GenBank已公布的Derf7核酸序列设计引物,用RT—PCR扩增获得其编码基因,插入pMD19-T载体进行序列测定和生物信息学分析。结果：获得Derf7全长基因约642bp,与参考序列（GenBankAY283292）同源性达99．7％％,仅在249位”A→G”和439位“C→T”发生点突变,含1个完整的开放读码框。推测编码蛋白由213个氨基酸组成,信号肽序列位于1-17aa,亲水性指数为0．031,跨膜区域位于171—190aa,二级结构由一螺旋（57．28％）、延伸主链（6．57％）和无规卷曲（36．15％）组成;亚细胞定位于细胞质,含有N-糖基化位点1个（151-154aa）,蛋白激酶c磷酸化位点1个（193-195aa）,酪蛋白激酶Ⅱ磷酸化位点2个（155—158aaand173．176aa）。N端酰基化位点1个（97—102aa）。粉尘螨和屋尘螨变应原第7组分氨基酸序列相似度为86％,二者在螨类第7组分氨基酸序列构建出的分子进化树中聚成一簇。结论：获得了Derf7全长基因,为进一步获得其基因工程制品用于临床和实验研究奠定了基础。     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.