Using cellular automata images and pseudo amino acid composition to predict protein subcellular location

Summary. The avalanche of newly found protein sequences in the post-genomic era has motivated and challenged us to develop an automated method that can rapidly and accurately predict the localization of an uncharacterized protein in cells because the knowledge thus obtained can greatly speed up the process in finding its biological functions. However, it is very difficult to establish such a desired predictor by acquiring the key statistical information buried in a pile of extremely complicated and highly variable sequences. In this paper, based on the concept of the pseudo amino acid composition (Chou, K. C. PROTEINS: Structure, Function, and Genetics, 2001, 43: 246–255), the approach of cellular automata image is introduced to cope with this problem. Many important features, which are originally hidden in the long amino acid sequences, can be clearly displayed through their cellular automata images. One of the remarkable merits by doing so is that many image recognition tools can be straightforwardly applied to the target aimed here. High success rates were observed through the self-consistency, jackknife, and independent dataset tests, respectively.     

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     

Sub-epitopic dissection of HCV E1<Subscript>315–328</Subscript>HRMAWDMMMNWSPT sequence by similarity analysis

Summary. Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1_315–328HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315–328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH₂ and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host’s proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment. Authors’ address: D. Kanduc, Department of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, Bari 70125, Italy     

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism

 The negative gravitropism of the sporangiophores of Phycomyces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132–142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15–25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains. Received: 2 September 1999 / Accepted: 9 November 1999     

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk

Summary. Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.     

Predicting DNA-binding proteins: approached from Chou’s pseudo amino acid composition and other specific sequence features

Summary. DNA-binding proteins play a pivotal role in gene regulation. It is vitally important to develop an automated and efficient method for timely identification of novel DNA-binding proteins. In this study, we proposed a method based on alone the primary sequences of proteins to predict the DNA-binding proteins. DNA-binding proteins were encoded by autocross-covariance transform, pseudo-amino acid composition, dipeptide composition, respectively and also the different combinations of the three encoded methods; further, these feature matrices were applied to support vector machine classifiers to predict the DNA-binding proteins. All modules were trained and validated by the jackknife cross-validation test. Through comparing the performance of these substituted modules, the best result was obtained from pseudo-amino acid composition with the overall accuracy of 96.6% and the sensitivity of 90.7%. The results suggest that it can efficiently predict the novel DNA-binding proteins only using the primary sequences. Authors’ address: Menglong Li, College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, P.R. China     

Extraordinary Conservation of Cysteines Among Homologous Chironomus Silk Proteins sp185 and sp220

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20–28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20–28 residues, 61% of which can be described by the motif X_5–8-Cys-X₅-(Trp/Phe/Tyr)-X₄-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5′- and 3′-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures. Received: 30 August 1996 / Accepted: 6 November 1996     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

Increased transglutaminase activity was associated with IL-6 release in cultured human gingival fibroblasts exposed to dental cast alloys

Summary. Molecular mechanisms underlying gingival and periodontal inflammation caused by dental alloys are still poorly understood. Recently, it has been demonstrated that tissue transglutaminase can be involved in inflammatory cell response. The aim of this study was to evaluate effects of exposure to orthodontic materials on transglutaminase in cultured human gingival fibroblasts. The incubation with Ni–Ti heat-activated (T3) or Ni–Ti super-elastic (T4), and with Ni–Cr–Co (T2) alloys produced respectively 2.5-fold and 8-fold increases in IL-6 release compared with control cultures. Transglutaminase activity was significantly increased in cells exposed to T3 and T4 alloys (about 170% of control; p < 0.05), where it was mainly localized close to inner part of cell membrane. The exposure to T3 and T4 specimens significantly up-regulated also tTG expression compared with control cultures. These data first show an association between IL-6 release and tissue transglutaminase increases, suggesting that TGase-mediated reactions may play a major role in periodontal inflammation.     

The α-gliadin gene family. II. DNA and protein sequence variation, subfamily structure, and origins of pseudogenes

 The derived amino-acid sequences of all reported α-gliadin clones are compared and analyzed, and the patterns of sequence change within the α-gliadin family are examined. The most variable sequences are two polyglutamine domains. These two domains are characteristic features of the α-gliadin storage proteins and account for most of the variation in protein size of this otherwise highly conserved protein family. In addition, their encoding DNA sequences form microsatellites. Single-base substitutions in the α-gliadin genes show a preponderance of transitions, including the C to T substitution which contributes to the generation of stop codons, and consequently to the observation that approximately 50% of the α-gliadin genes are pseudogenes. In one unusual gene, a microsatellite has expanded to 321 bp as compared to the normal 36–72 bp, and may result from similar mechanisms that produce polyglutamine-associated genetic diseases in humans. A comparison of the 27 reported sequences show several α-gliadin gene subfamilies, at least some of which are genome specific. Received: 1 October 1996/Accepted: 20 December 1996     

A Phylogenetic Assessment of the Eukaryotic Light-Harvesting Antenna Proteins, with Implications for Plastid Evolution

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment–protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin–chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin–chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes. Received: 25 February 1998 / Accepted: 13 May 1998     

Biotechnology applications of amino acids in protein purification and formulations

Summary. Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called “compatible solutes”, since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein–protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.     

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     

The inhibitory effect of various indolyl amino acid derivatives on arginase activity in macrophages

Summary. Numerous indolyl amino acids and their derivatives inhibited arginase activity. The inhibition was found to be non-competitive, – at least partly – allosteric, and independent on manganese ions in the active site, and it cannot be explained by the dissociation of arginase homotrimers. Indole alone is weakly inhibitory; however, the presence of three-carbon side chains and their net charges is favorable for the inhibition. The binding of the inhibitory compounds caused only minor changes in the steric structure of arginase: a slight increase in α-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and β-turn sections. A slight alteration in the tertiary structure was also found using tryptophane fluorescence studies, but buried apolar side chains were not transposed to the protein surface. Computer studies that were performed did not provide additional structural information. Authors’ address: András Hrabák, Department of Medical Chemistry, Molecular Biology and Patho-biochemistry, Semmelweis University Medical School, Budapest, VIII. Puskin u. 9., H-1444 POB 260, Hungary     

Phylogenetic Analysis of Three Lipocalin-Like Proteins Present in the Milk of Trichosurus vulpecula (Phalangeridae, Marsupialia)

Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents. Received: 28 October 1996 / Accepted: 19 May 1997     

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons

Summary. Nectins and Nectin-like molecules belong to the Ca-independent immunoglobulin superfamily of cell adhesion molecules and are mandatory for various cellular functions such as morphogenesis, differentiation and proliferation. Among them, Nectin-like molecule 1 (Necl-1) is unique for its exclusive expression in the brain where it is localized at the contact sites among axon terminals and glia cell processes, cooperatively forming synapses. We hereby aimed to unambiguously characterize Necl-1 at the protein level in rat brain. Rat cerebellar neurons were lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 5.96 with an observed molecular weight of 26 kDa was identified as Nectin-like molecule 1. MS/MS analyses of three matching peptides warranted unambiguous identification for the first time. Additionally, we verified the result by immunoblotting and detected two bands at about 48 kDa and 60 kDa. The proposed roles of Necl-1 in cerebellar morphogenesis as well as plasticity of synapses challenge further research on its function in more detail and we hereby provide a fair analytical tool for the unequivocal determination of Necl-1, independent of antibody availability and specificity.     

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     

Using string kernel to predict signal peptide cleavage site based on subsite coupling model

Summary. Owing to the importance of signal peptides for studying the molecular mechanisms of genetic diseases, reprogramming cells for gene therapy, and finding new drugs for healing a specific defect, it is in great demand to develop a fast and accurate method to identify the signal peptides. Introduction of the so-called {−3,−1, +1} coupling model (Chou, K. C.: Protein Engineering, 2001, 14–2, 75–79) has made it possible to take into account the coupling effect among some key subsites and hence can significantly enhance the prediction quality of peptide cleavage site. Based on the subsite coupling model, a kind of string kernels for protein sequence is introduced. Integrating the biologically relevant prior knowledge, the constructed string kernels can thus be used by any kernel-based method. A Support vector machines (SVM) is thus built to predict the cleavage site of signal peptides from the protein sequences. The current approach is compared with the classical weight matrix method. At small false positive ratios, our method outperforms the classical weight matrix method, indicating the current approach may at least serve as a powerful complemental tool to other existing methods for predicting the signal peptide cleavage site. The software that generated the results reported in this paper is available upon requirement, and will appear at http://www.pami.sjtu.edu.cn/wm. An erratum to this article is available at .     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000