Protein and Peptide Letters - Volume 13, Issue 5, 2006

Research in cellular regulation is an important area of study in drug development. This special issue highlights some of the recent advances in protein and peptide chemistry that is related to cellular regulation. The functional role of a protein molecule requires an understanding of its biologic activity involved in cells. The focus is on interpretation of structural information gathered through molecular biology and various techniques. Recent research has provided an abundance of new information on biochemistry of proteins and peptides. It is essential to update our current understanding of some protein structure and function to fully appreciate and apply these findings. This special issue describes some recent work on folding/unfolding of insulin and its structure and function relationship, and a recent progress on research into the integrin βA domain using maltose-binding protein as a fusion tag to prepare quantitative amount of soluble leukocyte integrin βA. This issue also includes a recent study on opioid receptor-like receptor, which utilizes both GOA and GOB for signal transduction. The receptor can utilize GaO variants to inhibit adenylyl cyclase and stimulate protein kinases in HEK293 cells. The study confirmed that the ORL1 receptor could interact with the two variants of GaO to inhibit AC and stimulate ERK1/2. The coupling to G0A/B sheds light on the molecular basis for some of the signaling properties of the ORL1 receptor, including the Ca2+ channels. In addition, site-directed mutagensis study of ferrochelatase of chironomidae showed a considerable difference from mammalian ferrochelatases in enzyme activity and metal binding with copper ions. The study identifies for the first time that the highly conserved H60 in ferrochelatase is a key molecular determinant in directing a catalytically mode of metal interaction in the active site. The functional roles of bovine pancreatic deoxyribonuclease I in the catalytic reaction and the contributions of N- and C-terminal domains in the enzyme folding are also presented. Vogel, etc. reports a study on the complex structures for many types of CaM-binding peptides and some target proteins and the versatility of CaM-target recognition. Leung's paper describes the application of mass spectrometry to the characterization of the signaling proteins in cells. Other studies include various chemical techniques such as x-ray crystallography, synthesis, sequence and proteome analysis to study the functional roles and stability of important proteins. This special issue would provide insights into the methodology and discovery of novel proteins and peptides and their functional roles in cells. It is difficult to detect distant protein family relationships and the presence of different domains by direct comparison of sequences. However, the functional roles of proteins in cells can be determined if the chemical structures of the proteins are known. These protein factors share similar signaling machinery that may change the duration and localization of signals in cells. The study has become critical as efforts proceed to decipher and manage them for beneficial effect. In conclusion, mapping the protein connections and providing useful information about signaling pathways is a challenging work. Using updated knowledge to decipher how proteins play functional roles in cells and change in connectivity to produce fine regulation will require the best tools we can have. Finally, we want to express our appreciation to authors of this special issue. Specially, we wish to thank PPL for giving us the opportunity and help in creating this special issue. We are also grateful to Ms Mandy Chan of CUHK for editorial assistance.

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15β (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin βA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the bA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.

The ORL1 receptors stably expressed in HEK 293 cells can utilize PTX-resistant mutants of GαoA/B to inhibit adenylyl cyclase (AC) and stimulate extracellular signal-regulated protein kinases (ERKs). However, development of AC superactivation and loss of ERK1/2 responsiveness induced by chronic activation of the ORL1 receptors remained PTXsensitive.

Site-directed mutagenesis study of the conserved residue in ferrochelatase of chironomidae showed the binding interaction of copper with histidine-60. The activities of the variants increase by > 4-fold with H60N and 2 fold with H60D. The study identifies for the first time that the highly conserved H60 is a key molecular determinant in directing a catalytically competent mode of metal binding in the active site.

Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.

Calmodulin (CaM) is a prototypical Ca2+-sensor protein that can control many important biological functions by binding to hundreds of target proteins. To gain insight into the versatility of CaM-target recognition, we have analyzed the complex structures for many types of CaM-binding peptides and some target proteins. In particular, some recently reported novel complex structures reveal that the versatile target binding of CaM is accommodated by its flexible domain arrangement and the malleability of its interfaces.

Analysis of changes in genes and protein profiles provides invaluable information to understand the activities and functions of proteins. However, their activities are further regulated by post-translational modifications, such as glycosylation and phosphorylation. Cell growth and apoptotic pathways are good examples of demonstrating the roles of phosphorylation in activation of protein cascades upon stimulation.

The formation of local structure, in short peptides has been probed by examining cleavage patterns and rates of proteolysis of designed sequences with a high tendency to form β-hairpin structures. Three model sequences which bear fluorescence donor and acceptor groups have been investigated: Dab-Gaba-Lys-Pro-Leu-Gly-Lys-Val-Xxx-Yyy-Glu-Val-Ala-Ala-Cys-Lys-NH2 ï EDANS Xxx-Yyy: Peptide 1=DPro-LPro, Peptide 2=DPro-Gly, Peptide 3=Leu-Ala Fluorescence resonance energy transfer (FRET) provides a convenient probe for peptide cleavage. MALDI mass spectrometry has been used to probe sites of cleavage and CD spectroscopy to access the overall backbone conformation using analog sequences, which lack strongly absorbing donor and acceptor groups. The proteases trypsin, subtilisin, collagenase, elastase, proteinase K and thermolysin were used for proteolysis and the rates of cleavage determined. Peptide 3 is the most susceptible to cleavage by all the enzymes except thermolysin, which cleaves all three peptides at comparable rates. Peptides 1 and 2 are completely resistant to the action of trypsin, suggesting that β-turn formation acts as a deterrent to proteolytic cleavage.

The HIV and SIV gp41 ectodomains are extremely stable to chemical and thermal denaturation and the observed stability has been proposed to be an important thermodynamic driving force for gp41-mediated fusion of the viral and target cell membranes. The importance of the disulphide bond and surrounding residues within the HIV gp41 loop have been assayed by DSC studies of wild type and mutant HIV gp41. Based on the thermal transition temperature, the disulphide bond and surrounding residues do not contribute to the thermal stability of gp41 and thus do not contribute to gp41-mediated membrane fusion.

Neutrophils constitute the first line of host defense against pathogens. In the present study 2-D gel electrophoresis- mass spectrometry technology was employed to analyze the human resting neutrophils proteome. One hundred and two conserved spots were subjected to peptide mass fingerprinting, yielding 22 identifications. Among the identified proteins, nine are related to the inflammatory process, two polypeptides are assigned to metabolic functions and five are classified as structural.

The structural class is an important feature in characterizing the overall topological folding type of a protein or the domains therein. Prediction of protein structural classification has attracted the attention and efforts from many investigators. In this paper a novel predictor, the AdaBoost Learner, was introduced to deal with this problem. The essence of the AdaBoost Learner is that a combination of many 'weak' learning algorithms, each performing just slightly better than a random guessing algorithm, will generate a 'strong' learning algorithm. Demonstration thru jackknife cross-validation on two working datasets constructed by previous investigators indicated that AdaBoost outperformed other predictors such as SVM (support vector machine), a powerful algorithm widely used in biological literatures. It has not escaped our notice that AdaBoost may hold a high potential for improving the quality in predicting the other protein features as well, such as subcellular location and receptor type, among many others. Or at the very least, it will play a complementary role to many of the existing algorithms in this regard.

A convenient and efficient method for the synthesis of dipeptidyl ureas and urea acids employing Osuccinimidyl-( 9H-fluoren-9-ylmethoxycarbonyl amino)methylcarbamates has been described. All the compounds, obtained in good yields, have been fully characterized by mass and NMR spectra.

Stepwise solid phase synthesis using the Fmoc chemistry is reported for a panel of 71-residue and novel unnatural chemokine analogs derived from vMIP-II. This demonstrates the feasibility of using this synthetic method to generate de novo designed protein ligand molecules to study the biology and pharmacology of chemokine receptors.

The quantitative analysis of metallochaperone proteins in biofluids (e.g. blood, liquor) may be a major prerequisite for clinical investigations concerning the structure-function relationships of biologically-active metal cofactorcontaining chaperones in protein-misfolding diseases (e.g. Alzheimer's or related diseases). For these purposes, a new state-of-the-art gel electrophoresis [quantitative preparative native continuous polyacrylamide gel electrophoresis procedure (QPNC-PAGE)] combined with biological mass and NMR spectrometries might essentially contribute to provide fundamental insights into the metabolisms of important metal cofactors in biological systems and the proper folding of metallochaperones in conformational diseases.

Escherichia coli clones expressing recombinant shiga toxin (Stx1) and its derivatives Stx1-A and Stx1-B subunits were established to release the proteins into periplasmic space. The expression was examined by SDS-PAGE to visualize the subunits. The secreted assembled subunits were extracted with polymyxin B and assessed for biological activity. The results showed that the presence of N-terminus leader sequence of the gene is essential for assembly of the subunits to yield biologically active holotoxin (AB5). The absence of the leader sequence did not affect the expression of the subunits but did disrupt the holotoxin assembly.

Periplasmic substrate binding proteins (PSBPs) are essential components of the bacterial periplasmic transport system, which transports a wide variety of nutrients from the periplasmic space to the cytoplasm. The glutamate/aspartate binding protein SfGlu/AspBP is a unique PSBP consisting of 279 amino acid residues. The SfGlu/AspBP gene was cloned, over-expressed, and purified by immobilized metal ion affinity chromatography and size-exclusion chromatography. The recombinant protein SfGlu/AspBP has been crystallized by the hanging-drop vapor-diffusion method and its Xray diffraction data were collected at an atomic resolution of 1.15 Å. The crystals belong to the space group P21 with unit cell parameters: a=48.41 Å, b=68.18 Å, c=80.21 Å and b = 98.78 . There are two molecules per asymmetric unit.

The present work analyzed the tetrameric stability of the hemoglobins from the rattlesnake C. durissus terrificus using analytical gel filtration chromatography, SAXS and osmotic stress. We show that the dissociation mechanism proposed for L. miliaris hemoglobin does not apply for these hemoglobins, which constitute stable tetramers even at low concentrations.

A halophilic nucleoside diphosphate kinase from a moderate halophile, Halomonas sp. 593 (593NDK), was found to be resistant to heat treatment, as indicated by the high level of activity recovery after heating at high temperatures. This is due to reversibility of thermal unfolding, not the high melting temperature, of the protein. The highly homologous NDK from non-halophilic organism, Pseudomonas aeruginosa, showed instability against heat treatment. Chimeric molecules consisting of each half of these two NDKs were constructed and characterized for their heat stability. The results showed that the N-terminal half of 593NDK contributes to the heat stability of the proteins. We discuss the possible reason for the observed difference in resistance to heat treatment between the 593NDK and PaNDK and between two chimeric proteins.

Hepatocyte nuclear factor 6 (HNF-6)/OC-1, a part of liver-enriched transcription factor, controls pancreas and liver development and regulates expression of several hepatic genes. DNA-binding region of HNF-6α bound to a 14-mer DNA fragment has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P2 with unit cell parameters of a = 73.0 Å, b = 39.0 Å, c = 106.5 Å, β= 107.6 . X-ray diffraction data were collected to 2.0 Å resolution.