Protein and Peptide Letters - Volume 21, Issue 5, 2014

Up-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships.

Tyrosine kinase inhibitors show great promise as clinical therapies, but small molecule inhibitors that are available in the clinic and under development bind to the adenosine triphosphate binding domain of the kinase, potentially limiting efficacy and selectivity. The development of antisense peptide inhibitors is a relatively unexplored area of research, and here we investigate inhibitory peptides specific for the Janus-associated kinase (JAK) family member, tyrosine kinase 2 (TYK2). We have developed peptides that are 2–3 times more selective for TYK2 than other JAK family members, with a TYK2 IC50 of 1.2 μM. In addition, TYK2 inhibitory peptides show specificity for TYK2-mediated functions over JAK1 functions in cell-based assays. These peptides provide a new tool for the development of specific peptide inhibitors for closely related tyrosine kinases.

AB204 is an Activin/BMP2 chimera, which has been found to exhibit a higher activity than Bone Morphogenetic Protein 2 (BMP2) in osteogenic activity. To prepare AB204 for its preclinical studies, AB204 has been characterized in various formulation buffers. We observed that AB204 purified by ion-exchange chromatography has low water solubility (2.0 mg/ml), whereas it has high water solubility (higher than 10.0 mg/ml) when purified by reverse-phase chromatography. Analysis of the purification procedures reveals that the buffer composition at the lyophilization step determines the solubility. Lyophilization from sodium acetate buffer at pH 4.5 resulted in formation of sodium hydroxide, which caused low solubility of AB204 by pH increase upon reconstitution in water. However, lyophilization from buffers, containing acetic acid or trifluoroacetic acid (TFA) rendered AB204 to be highly soluble. During the course of these analyses, we found a simple procedure to further reduce residual amount of TFA in the purified AB204.

Therapeutic potential of human acidic fibroblast growth factor (FGF1) resulting from its undeniable role in angiogenesis and wound healing processes is questioned due to its low stability and short half-life in vivo. Our previous studies showed that prolonged biological activity of FGF1 can be achieved by increasing its proteolytic resistance directly linked to improved global thermostability. In this study, we applied an alternative method of generation of long-lasting FGF1 variants by rigidification of the growth factor’s segment highly sensitive to proteases action. In order to determine regions the most prone to enzymatic degradation, we used limited proteolysis by trypsin combined with mass spectrometry analysis. We found that the initial proteolytic cleavages occurred mainly within the C-terminal region of the wild-type protein, pointing on its significant role in growth factor degradation. Based on bioinformatic analysis, we introduced two single mutations (C117P, K118V) within β-strand XI and combined them in a double mutant. We determined resistance to proteolysis, biophysical properties and biological activities of obtained variants. All of them occurred to be significantly less susceptible to trypsin (up to 100-fold) and also to chymotrypsin degradation comparing to the wild-type protein. Interestingly, all variants were not more thermostable than the wild-type FGF1. We attributed this dramatic increase in resistance to proteolysis to entropic stabilization of C-terminal region.

In mammalian cells, responses to hypoxia at the molecular transduction level are hallmarks of adaptation and survival under oxygen deprivation conditions. In this study, the protein expression patterns of mitogen-activated protein kinases (MAPKs) are investigated under hypoxia in primary cortical neurons and in a model of organotypic hippocampal slices in neonatal Sprague-Dawley rats. Abrupt fluctuations in MAPK expression can occur during anoxia, hypoxia, and relative hyperoxic shifts (e.g., reoxygenation); therefore, phosphorylation and dephosphorylation states could be crucial factors in metabolic reorganization for withstanding anaerobiosis. Whole cellular protein extracts were analyzed for the phosphorylation of MAPKpp38 and MAPKERK-1/2 (p44/p42) at threonine and tyrosine residues (Thr180/Tyr182) at different time periods of hypoxic exposure relative to a fixed normoxia control. The phospho-MAPKp38 (p-MAPKp38) to MAPKp38 relative unit ratio revealed that MAPKp38 expression increased in cortical neurons after 5 and 10 min, but decreased abruptly afterwards (20 – 120 min). The expression of phospho-MAPKERK-1 (p-MAPKERK-1/p44), however, decreased whereas that of p-MAPKERK-2/p42 increased compared to normoxia. In rat hippocampal slices (RHS), the expression of p-MAPKp38 was slightly but significantly higher in hypoxia, whereas the expression of p-MAPKERK-2/p42 increased and that of p-MAPKERK-1/p44 was intangible. This indicates that in cortical neurons hypoxia differentially upregulated the phosphorylation activation states of MAPKp38 and MAPKERK-1/2 (p44/p42), whereas in the RHS model MAPKp38 and MAPKERK-2/p42, but not MAPKERK-1/p44, phosphorylation states were upregulated in response to hypoxia. The neuroimmunological molecular patterns of the differential MAPK phosphorylation in vitro and ex vivo in response to hypoxic shift indicated a significant role for these kinases in cellular adaptation to oxygen deprivation, and thereby may identify physiologic and neuroprotective responsive signaling cofactors and pathways in cortical and hippocampal neurons during hypoxia.

Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.

Flooding stress restricts soybean growth, it results in decrease the production. In this report, to understand how nuclear proteins in soybean affected by flooding, abundance changes of those proteins was analyzed. Nuclear proteins were extracted from the root tips of soybean treated with or without flooding stress. The extracted proteins were analyzed using a label-free quantitative proteomic technique. Of a total of 94 nuclear proteins that were found to be responsive to flooding, the 19 and 75 proteins were increased and decreased, respectively. The identified flooding-responsive proteins were functionally classified, revealing that 8 increased proteins changed in protein synthesis, posttranslational modification, and protein degradation, while 34 decreased proteins were involved in transcription, RNA processing, DNA synthesis, and chromatin structure maintenance. Among these proteins, those whose levels changed more than 10 fold included two poly ADP-ribose polymerases and a novel G-domain-containing protein that might be involved in RNA binding. The mRNA expression levels of these three proteins indicated a similar tendency to their protein abundance changes. These results suggest that acceleration of protein poly-ADP-ribosylation and suppression of RNA metabolism may be involved in root tip of soybean under flooding stress.

Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical Km values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding within bacteria. TF possesses a three-state equilibrium in vivo: monomeric TF bound to ribosome, free monomeric, and dimeric TF in cytoplasm. TF consists of an N-terminal ribosome binding domain, a middle peptidyl–prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain involved in substrate binding and dimerization. Investigation of the effect of C-terminal 13 region on TF structure and function will help to further the understanding of its mechanism as a chaperone in vitro and in vivo. Here we present TF419, a TF mutant from which the C-terminal 13 residues were deleted to investigate the role of these residues in the structure stability and function of intact molecules. Small angle X-ray scattering (SAXS), fluorescence measurements and limited proteolysis results suggested that TF transitioned to a compact conformation when the Cterminal 13 residues were truncated. Further biochemical results reveal that TF dimerization was decreased as a result of the truncation. These results suggested that the C-terminal 13 residues play an important role in structural stability and chaperone function of TF.

Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase) is involved in the Mg2+-dependent hydrolysis of the phosphoenzyme at an optimum reaction temperature of 55°C. Bs-TpNPPase has been cloned and overexpressed in the E.coli M15 strain. Based on the conserved active sites, the protein was suggested to be a member of the haloalkanoate dehalogenase (HAD) superfamily. Two site-specific point mutants of Bs-TpNPPase were prepared by changing the catalytic Asp10 and Thr43 to Ala10 and Ala43, respectively. The activity of the two mutants further confirms Bs-TpNPPase as a member of the HAD superfamily. HAD superfamily can be divided into the four subfamilies and play several biochemical roles such as DNA repair, signal transduction and secondary metabolism. To understand the relationship between structure and thermostability in HAD superfamily, Bs-TpNPPase from Bacillus Stearothermophilus was selected. The X-ray crystal structure of Bs-TpNPPase was determined at 1.5A resolution using the molecular replacement phasing method. The structure of Bs-TpNPPase has been deposited and the PDB code is 4KN8. Compared with Bsp, a mesophilic prokaryotic putative p-nitrophenyl phosphatase from Bacillus Subtilis, Bs- TpNPPase showed highly homology but variations in the level of leucine content, aromatic clusters, cation-Pi and hydrophobic interaction. These differences may affect the thermal stability of the protein. The crystal structure of Bs-TpNPPase described herein may serve as a guide to better understand the mechanism of thermostability and provide insights for further mutation work.

Neuropeptide FF (NPFF) interacts with specific receptors to regulate diverse biological processes. Its modulatory effect in the immune field, however, has not been fully explored yet. Here, we report that NPFF2 receptors may be functionally expressed in two immune cell models, the primary peritoneal macrophage and RAW 264.7 macrophage. Firstly, the mRNA levels of NPFF2 receptor were up-regulated in macrophages when treated with LPS for 24 to 72 h. Subsequently, our data hinted that NPFF regulates the viability of both kinds of macrophages. After treatment with RF9, a reported antagonist for both NPFF receptors, delayed or inhibited the NPFF-induced macrophages viability augmentation, suggesting the involvement of NPFF2 receptor. Furthermore, down-regulation of nitric oxide (NO) synthases (NOSs) partially significantly inhibited the viability augmentation of macrophages induced by NPFF, implying a nitric oxide synthases- dependent pathway is involved. However, the NOSs are not the only route by which NPFF affects the viability of macrophages. Pharmacological inhibitors of NF-κB signal pathway also blocked the NPFF-induced macrophages growth, suggesting the involvement of the NF-κB signal pathway. The regulation activity of NPFF for macrophages suggests that NPFF could act as a potential hormone in the control of immune system. Collectively, our data provide new evidence about the immune modulatory effect of NPFF, which will be helpful in extending the scope of NPFF functions.

The cardiovascular heat shock protein (cvHsp/HspB7) exhibited cardiac-specific expression and is a possible candidate of dilated cardiomyopathy in heart failure. The molecular characteristics and biochemical properties of cvHsp are only partially understood. This study was aimed to identify the biological properties and molecular high-order structure of cvHsp. The cvHsp protein was prepared by the refined purification at large amount. The pooled fractions were existed as two types of oligomers in solution and exhibited chaperone-like activity. The circular dichroism analyzed ureainduced unfolding processes. Multiple sequence alignment and an automated protein modeling were used to describe the three-dimensional structural model of the cvHsp monomer and dimer. By the refined purification, the cvHsp appeared in oligomeric and dimeric forms (approximately 17 kDa and 40 kDa, respectively) composed of 18.6-kDa monomers. The cvHsp prevented dithiothreitol (DTT)-induced aggregation of the insulin B chain and conferred oligomeric unfolding process in urea-containing solution. It exhibited structural stability and conformed to the two-state folding/unfolding oligomerization model. According to sequence alignment of the rat cvHsp gene, three-dimensional model based on the crystallographic structure of wheat Hsp16.9 was reconstructed. The cvHsp presented two antiparallel β-sheet sandwich structure of sHsp’ core α-crystallin domain, and formed dimeric or oligomeric organization in solution. This work described the structural components of cvHsp and existed as the polydispersed molecular oligomers in vitro, which are some common properties of the sHsp family. These characteristics of the cvHsp gene is helpful to clarify molecular functionality in cardiac diseases.