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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.30, #4)

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use by E. Karnaukhova; Y. Ophir; B. Golding (pp. 317-332).

Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area¹.

Keywords: Keywords: Alpha-1-proteinase inhibitor – Antitrypsin – Emphysema – Glycosylation – Recombinant

Activity-based proteomics: enzymatic activity profiling in complex proteomes by H. Schmidinger; A. Hermetter; R. Birner-Gruenberger (pp. 333-350).

In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.

Keywords: Keywords: Activity-based proteomics – Functional proteomics – Enzymatic activity profiling – Active site-directed chemical probes

Strategies to improve plasma half life time of peptide and protein drugs by M. Werle; A. Bernkop-Schnürch (pp. 351-367).

Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half life times. By shortening the overall amino acid amount of somatostatin and replacing l-analogue amino acids with d-amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin. A PEG_2,40 K conjugate of INF-α-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.

Keywords: Keywords: Peptides – Proteins – Plasma half life time – N-C-terminus – PEGylation

Eye lens proteomics by W. Hoehenwarter; J. Klose; P. R. Jungblut (pp. 369-389).

The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.

Keywords: Keywords: Eye lens – Proteomics – Crystallins – 2-DE – Liquid chromatography – Mass spectrometry

Eye lens proteomics by W. Hoehenwarter; J. Klose; P. R. Jungblut (pp. 369-389).

Keywords: Keywords: Eye lens – Proteomics – Crystallins – 2-DE – Liquid chromatography – Mass spectrometry

Tuberin negatively affects BCL-2’s cell survival function by A. Freilinger; M. Rosner; M. Hengstschläger (pp. 391-396).

Uncontrolled cell cycle progression and cell growth are key properties of tumor cells. The tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC) have been demonstrated to control both, cell cycle and cell size regulation. Hamartin, encoded by TSC1, and tuberin, encoded by TSC2, form a complex, of which tuberin is assumed to be the functional component. Loss of TSC genes function triggers hamartoma development in TSC patients. However, in vivo mostly tumor cell development is rapidly terminated via apoptosis. BCL-2, the founding member of the BCL-2 family of proteins, is well known for its anti-apoptotic properties. Here we show that pro-apoptotic actinomycin D cannot interfere with BCL-2’s cell survival functions. However, we found tuberin to negatively regulate BCL-2’s anti-apoptotic effects on low serum-induced apoptosis. These findings warrant further investigations to elucidate the molecular mechanism underlying tuberin’s negative effects on cell survival.

Keywords: Keywords: Tuberous sclerosis – Tuberin – Hamartin – Cell survival – BCL-2

Classifying G protein-coupled receptors and nuclear receptors on the basis of protein power spectrum from fast Fourier transform by Y.-Z. Guo; M. Li; M. Lu; Z. Wen; K. Wang; G. Li; J. Wu (pp. 397-402).

As the potential drug targets, G-protein coupled receptors (GPCRs) and nuclear receptors (NRs) are the focuses in pharmaceutical research. It is of great practical significance to develop an automated and reliable method to facilitate the identification of novel receptors. In this study, a method of fast Fourier transform-based support vector machine was proposed to classify GPCRs and NRs from the hydrophobicity of proteins. The models for all the GPCR families and NR subfamilies were trained and validated using jackknife test and the results thus obtained are quite promising. Meanwhile, the performance of the method was evaluated on GPCR and NR independent datasets with good performance. The good results indicate the applicability of the method. Two web servers implementing the prediction are available at http://chem.scu.edu.cn/blast/Pred-GPCR and http://chem.scu.edu.cn/blast/Pred-NR.

Keywords: Keywords: G-protein coupled receptors – Nuclear receptors – Hydrophobicity – Fast Fourier transform – Power spectrum – Support vector machine

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes by Yu. A. Zolotarev; O. V. Dolotov; L. S. Inozemtseva; A. K. Dadayan; E. M. Dorokhova; L. A. Andreeva; L. Yu. Alfeeva; I. A. Grivennikov; N. F. Myasoedov (pp. 403-408).

Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.

Keywords: Keywords: ACTH(4-10) analog – Melanocortins – Degradation – HPLC – Glia – Neurons

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons by M. Gruber-Olipitz; J.-W. Yang; I. Slavc; G. Lubec (pp. 409-415).

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.

Keywords: Keywords: Nectin-like molecule 1 – Cerebellum – Neuron – Mass spectrometry – Proteomics

In vitro evaluation of the potential of thiomers for the nasal administration of Leu-enkephalin by A. Bernkop-Schnürch; K. Obermair; A. Greimel; T. F. Palmberger (pp. 417-423).

It was the aim of this study to evaluate the potential of thiolated polycarbophil for the nasal administration of Leucine-enkephalin (Leu-enkephalin). The enzymatic degradation of Leu-enkephalin on freshly excised bovine nasal mucosa was analysed qualitatively via thin layer chromatography and quantitatively via high performance liquid chromatography (HPLC). The potential of thiolated polycarbophil gels to provide a sustained release for the therapeutic peptide was investigated via diffusion studies. Permeation studies were performed in Ussing-type diffusion chambers with freshly excised bovine nasal mucosa. Results demonstrated that Leu-enkephalin is mainly degraded by the cleavage of tyrosine from the N-terminus of the peptide. Within one hour more than 63.5 ± 2% of this therapeutic peptide are degraded on the nasal mucosa. In the presence of 0.25% thiolated polycarbophil, this degradation process, however, could be significantly lowered. Diffusion studies demonstrated that Leu-enkephalin being incorporated in a 0.5% thiolated polycarbophil gel is sustained released out of it. The appearent permeability coefficient (P_app) for Leu-enkephalin on the nasal mucosa was determined to be 1.9 ± 1.2 × 10⁻⁷ cm/sec. Furthermore, in the presence of 0.5% thiolated polycarbophil and 1% glutathione, which is used as permeation mediator for the thiomer, the uptake of Leu-enkephalin from the nasal mucosa was even 82-fold improved. According to these results thiolated polycarbophil might be a promising excipient for nasal administration of Leu-enkephalin.

Keywords: Keywords: Leu-enkephalin – Nasal permeation – Enzyme inhibition – Thiomers – Thiolated polycarbophil

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose by D. J. S. Hinton; J. M. Ames (pp. 425-434).

We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N^ε-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N^ε-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.

Keywords: Keywords: Fructose – Advanced glycation endproduct – Site specificity – Fructoselysine – Heyns rearrangement products – N^ε-(carboxymethyl)lysine

Peptaibiomics: an advanced, rapid and selective analysis of peptaibiotics/peptaibols by SPE/LC-ES-MS by C. Krause; J. Kirschbaum; H. Brückner (pp. 435-443).

“Proteomics” and “peptidomics” are used as technical terms to define the analysis and study of all proteins and peptides expressed in an organism or tissue. In analogy we propose the name peptaibiomics for the analysis of a group of fungal peptide antibiotics (peptaibiotics) containing the characteristic amino acid Aib (α-aminoisobutyric acid). In analogy to the peptidome the complete expression of peptaibiotics by fungal multienzyme complexes should be named the peptaibiome.Peptaibiotics are defined as peptides containing Aib and exerting a variety of bioactivities. They comprise the sub-groups of N-acetylated peptaibols, characterized also by a C-terminal amide-linked 2-amino alcohol, and lipopeptaibols having in place of an acetyl group a lipophilic fatty acid acyl group. Furthermore, lipoaminopeptides are also known with long-chain fatty acid on the N-termini, a lipoamino acid in position three and a strongly basic secondary or tertiary amine form a subgroup of mixed forms which could not be integrated in one of these three previously mentioned groups.Here we present a specific and rapid screening method on the peptaibiome applicable directly onto filamentous fungi cultured in a single Petri dish. The method comprises solid-phase extraction (SPE) of peptaibiotics followed by on-line reversed-phase HPLC coupled to an ion trap electrospray tandem mass spectrometer (ES-MS). The presence of these peptides is indicated by characteristic mass differences of Δm = 85.1 Da representing Aib-residues which can be observed in the b-series of acylium fragment ions resulting from ES-MS. Partial sequences can be deduced from the data and compared with structures compiled in electronic peptaibol data bases. The judgement is possible whether or not structures are novel, already known or related to known structures. Suitability of the method is demonstrated with the analysis of strains of Trichoderma and its teleomorph Hypocrea. New sequences of peptaibiotics are presented and those being related to established 10- to 18-residue peptaibols trichovirin, trichogin and trichotoxin, which have been described in the literature.

Keywords: Keywords: α-Aminoisobutyric acid – Electrospray mass spectrometry – Fungal secondary metabolites – Peptide libraries – Solid-phase extraction

Extracellular matrix containing mutated fibrillin-1 (Fbn1) down regulates Col1a1, Col1a2, Col3a1, Col5a1, and Col5a2 mRNA levels in Tsk/+ and Tsk/Tsk embryonic fibroblasts by P. J. Christner; S. Ayitey (pp. 445-451).

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic fibroblast cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons 17–40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using β-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-β binding domains may (1) change the homeostasis of the ECM by binding additional growth factors and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.

Keywords: Keywords: Extracellular matrix – Fibrillin – Collagen – mRNA – Signaling – Scleroderma – Animal model – Mouse

Hybrid α/β-peptides: For-Met-Leu-Phe-OMe analogues containing geminally disubstituted β^2,2- and β^3,3-amino acids at the central position by A. Mollica; M. Paglialunga Paradisi; D. Torino; S. Spisani; G. Lucente (pp. 453-459).

The two fMLF-OMe analogues For-Met-β³hAc₆c-Phe-OMe (6) and For-Met-β²hAc₆c-Phe-OMe (12) and their corresponding N-Boc derivatives 5 and 11 have been synthesized and their biological activity towards human neutrophils evaluated. The N-formyl peptides 6 and 12 exhibit good activity as chemoattractans and 12 is highly active in superoxide anion production. The preferred solution conformation of the two N-formyl derivatives has been discussed.

Keywords: Keywords: Chemotactic peptides – Conformation – Hybrid α/β-peptides – Neutrophils

Prediction of protein homo-oligomer types by pseudo amino acid composition: Approached with an improved feature extraction and Naive Bayes Feature Fusion by S.-W. Zhang; Q. Pan; H.-C. Zhang; Z.-C. Shao; J.-Y. Shi (pp. 461-468).

The interaction of non-covalently bound monomeric protein subunits forms oligomers. The oligomeric proteins are superior to the monomers within the scope of functional evolution of biomacromolecules. Such complexes are involved in various biological processes, and play an important role. It is highly desirable to predict oligomer types automatically from their sequence. Here, based on the concept of pseudo amino acid composition, an improved feature extraction method of weighted auto-correlation function of amino acid residue index and Naive Bayes multi-feature fusion algorithm is proposed and applied to predict protein homo-oligomer types. We used the support vector machine (SVM) as base classifiers, in order to obtain better results. For example, the total accuracies of A, B, C, D and E sets based on this improved feature extraction method are 77.63, 77.16, 76.46, 76.70 and 75.06% respectively in the jackknife test, which are 6.39, 5.92, 5.22, 5.46 and 3.82% higher than that of G set based on conventional amino acid composition method with the same SVM. Comparing with Chou’s feature extraction method of incorporating quasi-sequence-order effect, our method can increase the total accuracy at a level of 3.51 to 1.01%. The total accuracy improves from 79.66 to 80.83% by using the Naive Bayes Feature Fusion algorithm. These results show: 1) The improved feature extraction method is effective and feasible, and the feature vectors based on this method may contain more protein quaternary structure information and appear to capture essential information about the composition and hydrophobicity of residues in the surface patches that buried in the interfaces of associated subunits; 2) Naive Bayes Feature Fusion algorithm and SVM can be referred as a powerful computational tool for predicting protein homo-oligomer types.

Keywords: Keywords: Naive Bayes Feature Fusion – Support vector machine – Pseudo amino acid composition – Weighted auto-correlation function – Homo-oligomer

Prediction of protein structural classes using support vector machines by X.-D. Sun; R.-B. Huang (pp. 469-475).

The support vector machine, a machine-learning method, is used to predict the four structural classes, i.e. mainly α, mainly β, α–β and fss, from the topology-level of CATH protein structure database. For the binary classification, any two structural classes which do not share any secondary structure such as α and β elements could be classified with as high as 90% accuracy. The accuracy, however, will decrease to less than 70% if the structural classes to be classified contain structure elements in common. Our study also shows that the dimensions of feature space 20² = 400 (for dipeptide) and 20³ = 8 000 (for tripeptide) give nearly the same prediction accuracy. Among these 4 structural classes, multi-class classification gives an overall accuracy of about 52%, indicating that the multi-class classification technique in support of vector machines may still need to be further improved in future investigation.

Keywords: Keywords: Support vector machines – CATH – Multi-class – Protein structural class prediction – Jackknifing

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy by J. W. Yang; T. Czech; M. Felizardo; C. Baumgartner; G. Lubec (pp. 477-493).

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.

Keywords: Keywords: Antioxidant – Chaperone – Cytoskeleton – Hippocampus – Mesial temporal lobe epilepsy (MTLE)

Identification of a glutathione S-transferase without affinity for glutathione sepharose in human kidney by T. Simic; M. Pljesa-Ercegovac; A. Savic-Radojevic; M. Hadziahmetovic; J. Mimic-Oka (pp. 495-498).

To identify kidney glutathione S-transferase (GST) isoenzyme, which does not bind to glutathione affinity column, biochemical characterization was performed by using an array of substrates and by measuring sensitivity to inhibitors. Immunological characterization was done by immunoblotting. Affinity flow-through GST exhibited activity towards 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and cumene hydroperoxide, typical class α substrates and high sensitivity towards hematin, an α class inhibitor. It cross-reacted with antibodies against α class GST. Affinity flow-through GST in human kidney is an α class member.

Keywords: Keywords: Glutathione S-transferase – Alpha class GST – Human kidney

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.30, #4)