Protein and Peptide Letters - Volume 22, Issue 3, 2015

Several studies have reported differences in physiological and pathological phenotypes between different strains of experimental mice, such as environment-based behavior, skin damage, damage in response to toxins and nervous system injury. However, the mechanisms underlying these differences have not yet been fully elucidated. We have been studying the function of kallikrein-related peptidases (KLKs), serine proteases known to serve a variety of functions. In this study, we focused on differences in KLKs between C57BL/6 mice and 129 mice. Among 13 KLKs genes examined, 12 KLKs showed differences in the mRNA coding region sequence and 7 KLKs showed different deduced amino acid sequences of their proteins when comparing C57BL/6 and 129 mice. KLK6 protein from 129 mice had six amino acid differences compared with that from C57BL/6 mice. KLK6 protein from 129 mice showed reduced SDS-PAGE mobility compared with that from C57BL/6 mice. Moreover, recombinant KLK6 protein from 129 mice had a higher optimum pH and >15 times higher hydrolytic enzymatic activity for several substrates than that from C57BL/6 mice. These results suggest that KLKs may contribute to the genetic basis of the differences between mouse strains.

The P2 protein (equivalent of L7/L12 in prokaryotes), a member of the ribosomal stalk in eukaryotes, is highly conserved, particularly its C-terminal domain. In order to understand the sequence-structure-function relationships in eukaryotic C-terminal stretches, about which nothing is known at the moment, we have investigated here, the structural characteristics of these domains of P2 proteins from three different species, namely, human, Plasmodium falciparum, and Toxoplasma gondii; the sequence homology among these is 70% although sequence identity is only 36%. About 50 amino acids of the C-terminal domains of P2 from the three species were expressed and purified. Gel filtration studies indicated peaks for both monomer and oligomer at milimolar concentrations and also suggested monomer-multimer equilibrium. Circular Dichroism showed that this domain does not have stable secondary structures. 1H-15N HSQC spectra in every case showed one set of requisite number of peaks as per the sequence. This indicated that there is rapid multimer-monomer equilibrium in solution and the observed peaks which originate from the monomer reflect average chemical shifts. The spectral dispersion in all the cases is narrow, although there are noticeable differences in the three proteins. Detailed NMR investigations revealed that this protein domain is intrinsically disordered although there are short segments with preferred secondary structural propensities at similar places along the sequence. This may suggest that the sequence is selected in evolution to impart disorder, and thereby accord conformational adaptability.

The Arabidopsis thylakoid membrane bimodular oxidoreductase, AtVKOR, could catalyze disulfide bond formation, and its direct functional domain (thioredoxin-like domain) is located in the thylakoid lumen according to the topological structure. Many proteins have one or several disulfide bonds in the thylakoid lumen, including photosynthetic chain components. A yeast two-hybrid assay was used to identify potential targets for the AtVKOR, and a Trx-like domain was constructed into a BD vector as bait. Twenty-two thylakoid lumenal proteins with disulfides were selected. The cDNAs encoding these proteins were constructed into an AD vector. Eight proteins were identified from the hybrid results to interact with AtVKOR, including HCF164, cytochrome c6A, violaxanthin deepoxidase, embryo sac development arrest 3 protein (EDA3), two members pentapeptide repeat proteins (TL17 and TL20.3), and two FK-506 binding proteins (FKBP13 and FKBP20-2). The BIACORE system was used to demonstrate that the recombinant HCF164 and Trx-like domain of AtVKOR could interact directly in vitro. The KD value for binding HCF164 to AtVKOR was calculated as 2.5×10-6 M. These results suggest that AtVKOR can interact with partial thylakoid lumenal proteins and indicates AtVKOR plays an important role in regulating the thylakoid lumen redox.

A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 Å. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH.

Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding, and many different peptide-proline cis-trans isomerases (PPIases) catalyze this reaction. The acceleration of isomerization would be beneficial for in vitro refolding of protein preparations for industrial and research purposes. So we analyzed whether low-molecular-weight compounds that have been reported to enhance protein refolding have the activity to accelerate the isomerization. To evaluate the effects of chemicals on the isomerization rate, we set up a new NMR (EXSY) method that is invulnerable to their inhibitory activity, if any, and to their large NMR signals. With this method, we found that dimethylbenzylammonium propane sulfonate (NDSB-256) increase the isomerization rate in a concentration-dependent manner for the first time. Acceleration by imidazole (suggested but not experimentally confirmed) was also demonstrated. Arginine, a most popular refolding additive, did not show any significant effects on the isomerization reaction as expected.

Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain–cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain– cystatin affinity.

Human C-reactive protein (CRP) is an acute phase protein, which harbours both host defence and scavenging properties. In this study, we obtained two new crystal forms of CRP, where CRP forms a symmetric, staggered dimer of pentamers. In one of these structures, obtained in the presence of HIV-1 Tat protein, this dimer of pentamers is stabilized by two zinc ions trapped within a cleft of the effector face of CRP. These two decameric interfaces involve complementary surfaces of CRP pentamers and bury a large area of ∼2000 Å2 per pentamer, suggesting a biological role of this interface. These two novel decameric interfaces and the involvement of zinc might have important consequences in the understanding of CRP biological functions.

Earlier peptidomic analysis of the skin secretion of Xenopus amieti led to the identification of orthologs of magainins and other peptides. This study investigated the degradation, in vitro insulin-releasing and acute metabolic effects of magainin-AM1 (GIKEFAHSLGKFG KAFVGGILNQ) and magainin–AM2 (GVSKILHSAGKFGKAFLGEIMKS). Plasma degradation was investigated using reversed-phase HPLC and MALDI-TOF mass spectroscopy. Insulin-releasing effects were determined using BRIN-BD11 clonal beta cells and mouse islets. Effects of magainin peptides on cytosolic enzyme lactate dehydrogenase release, membrane potential and intracellular Ca2+ concentration were assessed using BRIN-BD11 cells while their in vivo effects on glucose tolerance and insulin release were assessed in obese, insulin-resistant Swiss National Institute of Health (NIH) mice. Both peptides were resistant to degradation by plasma enzymes in vitro for up to 8 h. Though magainin-AM1 elicited non-toxic, concentration-dependent stimulation of insulin-release from clonal BRINBD11 cells at concentrations ≥ 100nM, magainin-AM2 produced a higher stimulation of insulin-release from BRIN-BD11 cells and isolated mouse islets. Membrane depolarization and intracellular [Ca2+]i in BRIN-BD11 cells were significantly (P<0.05) induced by both peptides and chelation of extracellular Ca2+, addition of diazoxide or verapamil significantly (P<0.01) reduced the insulinotropic actions of the peptides. Administration of magainin-AM2 (75 nmol/kg body weight) to high-fat fed mice significantly enhanced insulin-release (P<0.01) and improved glucose tolerance (P<0.05). These data indicate magainin-AM2 peptides have potential for development into agents for treatment of type 2 diabetes.

We have developed a method to align a pair of EF-hand domains, an EF-lobe, using the symmetry axis that is intrinsic to the EF-lobe itself. The coordinate system for the alignment is dependent only on the symmetry of the EF-lobe. The absolute positions of each component in the structure can be described in this coordinate system. Our site provides the foundation for the analyses and comparisons of structural features of EFlobes.

Neuropeptide FF (NPFF) has been implicated in many physiological processes. Previously, we have reported that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. In this study, we investigated the influence of NPFF on lipopolysaccharide (LPS)-mediated osteoclast formation of RAW264.7 cells. Our results suggest that, NPFF dose-dependently (1 nM, 10 nM and 100 nM) inhibited osteoclast formation, TRAP enzyme activity and bone resorption in osteoclasts induced by LPS respectively. Moreover, LPS-provoked NO release was also inhibited by NPFF treatment, indicating a NO-dependent pathway is mainly involved. Furthermore, the alterations of osteoclast marker genes were also assessed including TRAP, Cathepsin K, MMP-9, NFATc1 and Runx2. NPFF downregulated LPS-caused gene augmentations of TRAP, Cathepsin K and MMP-9, whereas showed no influences on NFATc1 and Runx2. In addition, NPFF receptor 2 (NPFFR2) mRNA expression was also augmented in response to NPFF treatment, hinting the involvement of NPFFR2 pathway. It should be mentioned that RF9 (1 µ M), a reported pharmacological inhibitor for NPFF receptors, exerted NPFF-like agonist properties as to attenuate osteoclastogenesis. Collectively, our findings provide new evidence for the in vitro activity of NPFF on osteoclasts, which may be helpful to extend the scope of NPFF functions.

High temperature stress, especially on the early season of plant growth stages, is an agricultural problem in many areas in the world. A temporary or continually high temperature leads to a set of morphological, biochemical and physiological changes in plants, which consequently reduces the plant growth and development and finally may cause a severe reduction in economic yield. The main goal of this study was to assess plant response to high temperature stress (HTS) in early seedling of canola. This study is the first experiment on the effect of heat stress on proteome of canola. In the present research, a proteomics approach was used to evaluate the effects of high temperature stress, including 45 °C day/34 °C night for 2, 6 and 12 hour, on early seedling stage (2-day old) of canola. Proteins were isolated from hypocotyl and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 381 protein spots, 28 and 34 proteins were significantly down- and up-regulated, respectively. The trend of mRNA expression for sucrose binding protein, a scorbate peroxidase and triosephosphateisomerase, was in accordance with their trend at translation level. Results of this study suggest that the up-regulation of proteins involved in cellular traffic, energy and metabolism, and down-regulation of some proteins involved in disease and defense, protein synthesis and signal transduction could be the main reason of physiological and morphological responses to high temperature stress. The observed increases in the level of ascorbate peroxidase protein and mRNA expression in canola hypocotyl in response to HTS suggests that ascorbate peroxidase is a short term high temperature stress response protein and is thus a candidate for gene modification strategies aimed at producing high temperature canola varieties. These results also suggest that the up regulation of protein involved in energy and metabolism in response to the heat stress can use most of nutritive reserves in seedling of canola and might explain the reduced growth of canola in heat stress conditions.