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Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1-2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated "GE" motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The differential diagnostic role of plasma developmental endothelial locus-1 (Del-1) was proposed in our previous study. Therefore the current study aimed to confirm the diagnostic role and explore the prognostic role of exosomal Del-1 in a prospective cohort of female patients with breast cancer., Patients and Methods: To determine the optimal sampling time for the postoperative Del-1 measurements, blood was serially collected on days 1, 3, 5, and 7 after surgery in 22 patients (cohort 1). Thereafter, 111 female patients with breast cancer were prospectively enrolled (cohort 2) to compare exosomal Del-1 levels before and after surgery., Results: Among the subsequent prospective cohort, 107 patients (96.4%) showed a high exosomal Del-1 level (optical density [OD] value > 0.5) at the time of diagnosis. Of these patients, 101 (94.6%) in this high-level group showed normalized Del-1 levels postoperatively, representing a significant difference (mean OD value, 1.232 vs. 0.196; P < .00001). High postoperative Del-1 level was significantly associated with a worse disease-free survival adjusted to the clinicopathological characteristics (hazard ratio, 24.0; P = .0011)., Conclusion: This study confirmed the normalization of exosomal Del-1 after surgery, indicating exosomal Del-1 as a potent diagnostic biomarker for breast cancer. In addition, because a high Del-1 level after surgery was associated with early relapse, this suggests exosomal Del-1 as a potential prognostic marker by identifying the existence of residual cancer., Competing Interests: Disclosure This study was supported by the National Research Foundation. The authors have stated that they have no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration.

Background: Few reports of Echinococcus spp. have been described in the USA; however, the geographical distribution of Echinococcus spp. in wild hosts is increasing consequent to human activities. In the early 2000's, 253 elk (Cervus canadensis) originating from Alberta, Canada were released into the Great Smoky Mountains National Park and North Cumberland Wildlife Management Area in an effort to re-establish their historical range., Methods: We investigated the prevalence of Echinococcus spp. in re-established elk populations in the North Cumberland Wildlife Management Area and the Great Smoky Mountains National Park via a retrospective analysis of banked elk tissues and helminth examinations on intestinal contents from coyotes (Canis latrans) from the North Cumberland Wildlife Management Area., Results: Four elk were PCR and sequence positive for E. canadensis. Each sequence had 98% or greater coverage and identity to multiple E. canadensis genotypes on GenBank. Adult Echinococcus spp. were not detected in any of the coyotes examined in this study., Conclusions: Continued surveillance of this disease in susceptible species in these areas is warranted, and these data further underscore the risk of zoonotic pathogen introduction secondary to wildlife translocation.

Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity ( i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo , which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high K m for O 2 suggested that O 2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases., (© 2020 Brewitz et al.)

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD 100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD 100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD 100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Quality control of newly synthesized glycoproteins is tightly regulated by sugar processing of N-glycans and by recognition of specific glycan structures by lectins in the endoplasmic reticulum (ER). Mannose trimming and its recognition determine the targeting of misfolded glycoproteins for ER-associated degradation. ER degradation-enhancing α-mannosidase-like (EDEM) proteins in mammals and their homologue Htm1p/Mnl1p in Saccharomyces cerevisiae are involved in this process. To analyze the function of EDEM proteins, we expressed and purified recombinant EDEM3 from HEK293 cells and assessed its mannose-trimming activity in vitro.

Regucalcin plays a pivotal role as a suppressor of human carcinogenesis, and downregulation of regucalcin expression may contribute to the promotion of human osteosarcoma. Overexpression of regucalcin suppressed the proliferation of Saos-2 human osteosarcoma cells in vitro and decreased the protein levels of multiple signaling components, transcription factors, and tumor suppressors. Interestingly, extracellular regucalcin repressed colony formation and proliferation of Saos-2 cells, and reduced the protein levels of multiple signaling components, cell cycle inhibitor, and various transcription factors. Thus, regucalcin suppressed the growth of human osteosarcoma cells, providing a novel strategy with the gene therapy for treatment of osteosarcoma.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

The investigation of the interacting proteins with testis-specific calcium-binding protein CBP86-IV (CABYR) was carried out in human spermatozoa. The total RNA from human spermatozoa was extracted, and the ORF sequence of TSCBP86-IV gene was amplified and cloned into expression vector pET-28a. The positive recombinant clones were transformed into Escherichia coli strain BL21 (DE3) to express fusion protein. Then, co-immunoprecipitation (Co-IP) of TSCBP86-IV was performed in BL21 cell lysate expressing CBP86-IV recombinant protein. The immune complex was captured and identified by mass spectrometry. Reverse Co-IP of potential interacting proteins was performed in human sperm cell lysate. The potential protein interactions were confirmed by yeast two-hybrid system. Thirteen proteins were successfully identified in immune complex from E. coli cell lysate. Phosphoglycerate kinase 2 (PGK2) further showed positive results both in reverse Co-IP and yeast two-hybrid experiments and was confirmed to be interacted with TSCBP86-IV in human sperm cells., (© 2019 Blackwell Verlag GmbH.)

Introduction: Amaranthus retroflexus (Redroot Pigweed) is one of the main sources of allergenic pollens in temperate areas. Polcalcin is a well-known panallergen involved in cross-reactivity between different plants. The aim of this study was the molecular cloning and expression of polcalcin, as well as evaluating its IgE-reactivity with A. retroflexus sensitive patients' sera., Methods: Allergenic extract was prepared from A. retroflexus pollen and the IgE-reactivity profile was determined by ELISA and immunoblotting using sera from twenty A. retroflexus sensitive patients. Polcalcin-coding sequence was amplified by conventional PCR method and the product was inserted into pET-21b(+) vector. The recombinant protein was expressed in E. coli BL21 and purified by metal affinity chromatography. The IgE-binding capability of the recombinant protein was analyzed by ELISA and immunoblotting assays, and compared with crude extract., Results: Of 20 skin prick test positive patients, 17 patients were positive in IgE-specific ELISA. Western blotting confirmed that approximately 53% of ELISA positive patients reacted with 10kDa protein in crude extract. The A. retroflexus polcalcin gene, encoding to 80 amino acid residues was cloned and expressed as a soluble protein and designated as Ama r 3. The recombinant polcalcin showed rather identical IgE-reactivity in ELISA and western blotting with 10kDa protein in crude extract. These results were confirmed by inhibition methods, too., Conclusion: The recombinant form of A. retroflexus polcalcin (Ama r 3) could be easily produced in E. coli in a soluble form and shows rather similar IgE-reactivity with its natural counterpart., (Copyright © 2019 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Purification and separation of calpains and calpastatin are used to determine the individual activities of calpain-1 and calpain-2 and their inhibitor calpastatin. We discuss here a method to purify these enzymes using dialysis followed by separation using anion-exchange chromatography coupled with gradient elution. Swollen DEAE Sephacel is used as the column matrix in this method. Calpastatin and both domains of calpain are weakly basic molecules that effectively bind with the DEAE Sephacel and separate well using a stepwise, increasing gradient of NaCl to elute the proteins. Calpastatin binds most weakly with the column matrix, so it elutes first, followed by calpain-1 and, finally, calpain-2.

The production of recombinant calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific calpastatin species that can be present concomitantly with other calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various calpastatin species for several reasons among which: calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other calpastatin species deriving from different tissues or organisms and calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/calpastatin system in vitro.

We here describe the purification of calpastatin from human erythrocytes. When calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca 2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying calpastatin inhibition.

The Na + /H + exchanger NHE1 is critical for cell vitality as it controls intracellular pH and cell volume. Its functionality is influenced by calcineurin B homologous proteins (CHPs). The human isoform CHP3 is important for transport of NHE1 to the plasma membrane and for its activity. Here, we characterized the binding interaction of human CHP3 with the regulatory domain of NHE1. The exact binding site of CHP3 was previously debated. CHP3 as well as both regions of NHE1 in question were produced and purified. CHP3 specifically formed stable complexes with the CHP-binding region (CBD) of NHE1 (residues 503-545) in size-exclusion chromatography (SEC), but not with the C-terminal region (CTD, residues 633-815). CTD was functional as shown by Ca 2+ -dependent binding of calmodulin in SEC analysis. CHP3 bound with high affinity to CBD with an equilibrium dissociation constant (K D ) of 56 nM determined by microscale thermophoresis. The high affinity was substantiated by isothermal calorimetry analysis (K D  = 3 nM), which also revealed that the interaction with CBD is strongly exothermic (ΔG° = -48.6 kJ/mol, ΔH = -75.3 kJ/mol, -TΔS° = 26.7 kJ/mol). The data provide insights in the molecular mechanisms that underlie the regulatory interaction of CHP3 and NHE1 and more general of calcineurin homologous proteins with their target proteins.

Calcium binding peptides from Pacific cod ( Gadus macrocephalus ) bone have attracted attention due to their potential effects on bone health. In this study, calcium binding peptides (CBP) were prepared from Pacific cod bone by trypsin and neutral protease. Ultraviolet spectra, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) revealed that carboxyl and amino groups in CBP could bind to Ca 2+ , and form the peptide-calcium complex (CBP-Ca). Single-pass intestinal perfusion (SPIP) experiments indicated that the intestinal calcium absorption was significantly enhanced ( p < 0.01) in CBP-Ca treated Wistar rats. The anti-osteoporosis activity of CBP-Ca was investigated in the ovariectomized (OVX) Wistar rat model. The administration of CBP-Ca significantly ( p < 0.01) improved the calcium bioavailability, trabecular bone structure, bone biomechanical properties, bone mineral density, and bone mineralization degree. CBP-Ca notably ( p < 0.01) increased serum calcium, however, it remarkably ( p < 0.01) reduced the levels of osteocalcin (OCN), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase isoform 5b (TRAP5b), and C-telopeptide of type I collagen (CTX-1) in serum. Results suggested that the cod bone derived CBP could bind with calcium, improve the intestinal calcium absorption, calcium bioavailability, and serum calcium, then reduce the bone turnover rate, and thus ameliorate osteoporosis.

In Tetrahymena, K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena. A 40kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Staphylococcus aureus cell wall anchored Serine Aspartate repeat containing protein D (SdrD) is a member of the microbial surface component recognising adhesive matrix molecules (MSCRAMMs). It is involved in the bacterial adhesion and virulence. However the extent of genetic variation in S. aureus sdrD gene within isolates from healthy carriers are not known. The aim of this study was to evaluate allelic variation of the sdrD gene among S. aureus from healthy nasal carriers., Results: The sdrD A region from 48 S. aureus isolates from healthy carriers were analysed and classified into seven variants. Variations in the sdrD A region were concentrated in the N2 and N3 subdomains. Sequence analysis of the entire sdrD gene of representative isolates revealed variations in the SD repeat and the EF motifs of the B repeat. In silico structural modelling indicates that there are no differences in the SdrD structure of the 7 variants. Variable amino acid residues mapped onto the 3D structure revealed that the variations are surface located, exist within the groove between the N2-N3 subdomains and distributed mainly on the N3 subdomain. Comparison of adhesion to keratinocytes in an in vitro cell adhesion assay, using NCTC 8325-4∆sdrD strains expressing the various sdrD gene variants, indicated a significant difference between only two complements while others showed no major difference in their adhesion., Conclusions: This study provides evidence of sequence variations across the different domains of SdrD from S. aureus isolated from healthy nasal carriers. Proper understanding of these variations is necessary in the study of S. aureus pathogenesis.

Trypsin was used for preparing peptides with high calcium-binding capacity from Antarctic krill. Hydroxyapatite chromatography (HAC), size-exclusion chromatography (SEC), and reversed phase high performance liquid chromatography (RP-HPLC) were used to capture and purify calcium-binding peptides. The peptide sequence was determined to be VLGYIQIR (N- to C-terminal, MW = 960.58 Da), using LTQ Orbitrap XL. According to the results of FTIR and mass spectrometry, chelating site of calcium ions may possibly involve the carbonal or amino groups of Gln, Ile and Arg residues. Molecular dynamic simulation showed the conformation of peptide was markedly varied, and the distance between calcium ion and Gln and Ile residues was changing all the time. However, the distance between calcium ion and carboxyl oxygen of arginine residues was not changed significantly from 2 ns to 100 ns. Identified peptide can be used as a novel calcium supplement., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Functional studies of extracellular proteins are often performed using coimmunoprecipitation without purified proteins. However, in order to exclude unspecific reactions of contaminants and for quantitative analysis of specific functions, it is necessary to use purified proteins. It is usually very difficult, however, to purify sufficient amounts of reasonably pure extracellular matrix proteins from tissue samples, but the recombinant expression and purification of proteins in eukaryotic expression systems including insect cells and mammalian cells has proven an alternative powerful method. In this chapter, we describe the expression and purification of recombinant fibulins, but the methods can be also used for other extracellular proteins., (© 2018 Elsevier Inc. All rights reserved.)

Regucalcin (RGN) is a calcium-regulating, anti-apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real-time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34-kDa, 28-kDa and 24-kDa isoforms, wherein the 34-kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium-related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments., (© 2017 Blackwell Verlag GmbH.)

Regucalcin is a calcium regulating multifunctional protein reported to have many important functions like calcium homeostasis, anti-oxidative, anti-apoptotic and anti-cancerous functions. Although it is demonstrated as a calcium regulating protein, the calcium binding ability of regucalcin is still a controversy. The main reason for the controversy is that it lacks a typical EF hand motif which is common to most of the calcium binding proteins. Even though many studies reported regucalcin as a calcium binding protein, there are some studies reporting regucalcin as non-calcium binding also. In the present study, we investigated the calcium binding ability of recombinant buffalo regucalcin by assessing the secondary structural changes of the protein using circular dichroism spectroscopy after adding Ca 2+ to the protein solution. Two types of calcium binding studies were done, one with different concentration of calcium chloride (0.5 mM CaCl 2 , 1 mM CaCl 2 , 2 mM CaCl 2 ) and other at different time interval (no incubation and 10 min incubation) after addition of calcium chloride. Significant structural changes were observed in both studies which prove the calcium binding ability of recombinant regucalcin. A constant increase in the α-helix (1.1% with 0.5 mM CaCl 2 , 1.4% with 1 mM CaCl 2 , 3.5% with 2 mM CaCl 2 ) and a decrease in β-sheets (78.5% with 0.5 mM CaCl 2 , 77.4% with 1 mM CaCl 2 , 75.7% with 2 mM CaCl 2 ) were observed with the increase in calcium chloride concentration. There was a rapid increase in α-helix and decrease in β-sheets immediately after addition of calcium chloride, which subsides after 10 min incubation.

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a major pest of rice in Asia, is able to successfully puncture sieve tubes in rice with its piercing stylet and then to ingest phloem sap. How BPH manages to continuously feed on rice remains unclear. Here, we cloned the gene NlSEF1, which is highly expressed in the salivary glands of BPH. The NlSEF1 protein has EF-hand Ca 2+ -binding activity and can be secreted into rice plants when BPH feed. Infestation of rice by BPH nymphs whose NlSEF1 was knocked down elicited higher levels of Ca 2+ and H 2 O 2 but not jasmonic acid, jasmonoyl-isoleucine (JA-Ile) and SA in rice than did infestation by control nymphs; Consistently, wounding plus the recombination protein NlSEF1 suppressed the production of H 2 O 2 in rice. Bioassays revealed that NlSEF1-knockdown BPH nymphs had a higher mortality rate and lower feeding capacity on rice than control nymphs. These results indicate that the salivary protein in BPH, NlSEF1, functions as an effector and plays important roles in interactions between BPH and rice by mediating the plant's defense responses.

Nesfatin-1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short-term and long-term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin-1 were cloned into a pET28a vector after the hexa-histidine-tagged multiple cloning sites sequence with an enterokinase recognition site incorporated in-between. The recombinant plasmid was transformed into Escherichia coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins. After purified by Ni-sepharose affinity chromatography, the fusion protein was treated with enterokinase to release nesfatin-1. The nesfatin-1 sample was further purified with reverse-phase high performance liquid chromatography (HPLC), and its molecular weight was determined by mass spectrometry. The biological activities of recombinant nesfatin-1 were also assessed using in vivo animal models. The method described here promises to produce about 8 mg biologically active nesfatin-1 with homogeneity over 98% from 1-L shaking flask culture of E. coli, which can be considered as an easy and cost-effective way to synthesize nesfatin-1., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

FH22 has been previously identified as a calcium-binding protein from the common liver fluke, Fasciola hepatica. It is part of a family of at least four proteins in this organism which combine an EF-hand containing N-terminal domain with a C-terminal dynein light chain-like domain. Here we report further biochemical properties of FH22, which we propose should be renamed FhCaBP1 for consistency with other family members. Molecular modelling predicted that the two domains are linked by a flexible region and that the second EF-hand in the N-terminal domain is most likely the calcium ion binding site. Native gel electrophoresis demonstrated that the protein binds both calcium and manganese ions, but not cadmium, magnesium, strontium, barium, cobalt, copper(II), iron (II), nickel, zinc, lead or potassium ions. Calcium ion binding alters the conformation of the protein and increases its stability towards thermal denaturation. FhCaBP1 is a dimer in solution and calcium ions have no detectable effect on the protein's ability to dimerise. FhCaBP1 binds to the calmodulin antagonists trifluoperazine and chlorpromazine. Overall, the FhCaBP1's biochemical properties are most similar to FhCaBP2 a fact consistent with the close sequence and predicted structural similarity between the two proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: A Disintegrin and Metalloproteinase 12 (ADAM12) is expressed significantly higher in multiple tumors than in normal tissues and has been used as a prognostic marker for the evaluation of cancer progression. Although several ADAM12 substrates have been identified biochemically and its proteolytic function has been explored, the upstream regulators and the interacting proteins have not been systematically investigated. Here, we use immunoprecipitation and mass spectrometry (MS)-based quantitative proteomic approaches to identify 28 interacting partners for the long form of ADAM12 (ADAM12-L) in HeLa cells. Proteins that regulate cell proliferation, invasion, and epithelial to mesenchymal transition are among the identified ADAM12-interacting proteins. Further biochemical experiments discover that the protein level and the stability of ADAM12 are upregulated by one of its interacting proteins, myoferlin. In addition, myoferlin also increases the proteolytic activity of ADAM12, leading to the reduction of an ADAM12 substrate, E-cadherin. This result implies that ADAM12 and its interacting proteins might converge to certain signaling pathways in the regulation of cancer cell progression. The information obtained here might be useful in the development of new strategies for modulating cell proliferation and invasion involved in the regulation between ADAM12 and its interacting partners. MS data are available via ProteomeXchange with identifier PXD003560., Biological Significance: Regulation of the proliferation and invasion of cancer cells is important in cancer treatment. ADAM12 has been found to play important roles in regulating these processes and identification of its interacting partners will improve our understanding of its biological functions and provide basis for functional modulation. Through mass spectrometry-based quantitative proteomic approaches, we identify the interacting partners for ADAM12 in a human cancer cell line and find many proteins that are involved in the proliferation and invasion of cancer cells. A novel regulator, myoferlin, of ADAM12 is discovered and this protein increases ADAM12 expression level, stability, and its enzymatic activity, leading to the reduction of its substrate, E-cadherin, which plays important roles in the regulation of cell adhesion and tumor metastasis. This result provides a connection for two highly expressed proteins in cancer cells and may shed light on the regulation of their biological functions in cancer progression., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.

Caleosins are a class of Ca(2+) binding proteins that appear to be ubiquitous in plants. Some of the main proteins embedded in the lipid monolayer of lipid droplets, caleosins, play critical roles in the degradation of storage lipids during germination and in lipid trafficking. Some of them have been shown to have histidine-dependent peroxygenase activity, which is believed to participate in stress responses in Arabidopsis. In the model plant Arabidopsis thaliana, caleosins have been examined extensively. However, little is known on a genome-wide scale about these proteins in other members of the Brassicaceae. In this study, 51 caleosins in Brassica plants and Arabidopsis lyrata were investigated and analyzed in silico. Among them, 31 caleosins, including 7 in A. lyrata, 11 in Brassica oleracea and 13 in Brassica napus, are herein identified for the first time. Segmental duplication was the main form of gene expansion. Alignment, motif and phylogenetic analyses showed that Brassica caleosins belong to either the H-family or the L-family with different motif structures and physicochemical properties. Our findings strongly suggest that L-caleosins are evolved from H-caleosins. Predicted phosphorylation sites were differentially conserved in H-caleosin and L-caleosins, respectively. 'RY-repeat' elements and phytohormone-related cis-elements were identified in different caleosins, which suggest diverse physiological functions. Gene structure analysis indicated that most caleosins (38 out of 44) contained six exons and five introns and their intron phases were highly conserved. Structurally integrated caleosins, such as BrCLO3-3 and BrCLO4-2, showed high expression levels and may have important roles. Some caleosins, such as BrCLO2 and BoCLO8-2, lost motifs of the calcium binding domain, proline knot, potential phosphorylation sites and haem-binding sites. Combined with their low expression, it is suggested that these caleosins may have lost function.

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Regucalcin serves a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. The regucalcin gene, which is localized on the X chromosome, consists of seven exons and six introns. Reductions in the gene expression of regucalcin have been suggested to serve a role in hepatocarcinogenesis in animal models and human patients, indicating a potential role as a suppressor protein in cancer. The aim of the current study was to investigate the effect of exogenous regucalcin on cell proliferation in the cloned human breast cancer MDA‑MB‑231 bone metastatic cell line in vitro. The proliferation of MDA‑MB‑231 cells was suppressed following the addition of regucalcin (0.1‑10 nM) in vitro. The suppression of proliferation was not enhanced in the presence of tumor necrosis factor‑α, PD98059, staurosporine, Bay K8644, wortmannin, 5,6‑dichloro‑1‑β‑D‑ribofuranosylbenzimidazole or gemcitabine. Exogenous regucalcin did not induce cell death in MDA‑MB‑231 cells in vitro. These data suggest that exogenous regucalcin possesses suppressive effects on the proliferation of human breast cancer MDA‑MB‑231 bone metastatic cells, and that this effect may be mediated through various intracellular signaling pathways in vitro. Exogenous regucalcin is suggested to function as a suppressor in cancer cell proliferation.

Purpose: Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers., Experimental Design: Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples., Results: We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2-10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting., Conclusions and Clinical Relevance: We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca(2+) concentration, which is a fundamental prerequisite for any Ca(2+)-based signalling system, is accomplished by complex mechanisms including Ca(2+) binding proteins acting as Ca(2+) buffers. In this work we investigated the occurrence of Ca(2+) binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus., Results: A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca(2+)-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca(2+)-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca(2+) binding ability of the M. loti protein was demonstrated by (45)Ca(2+)-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca(2+) adducts., Conclusions: The present data indicate that ferredoxin II is a major Ca(2+) binding protein in M. loti that may participate in Ca(2+) homeostasis and suggest an evolutionarily ancient origin for protein-based Ca(2+) regulatory systems.

Calreticulin (CRT) is a molecular chaperone with a molecular mass of 46 kDa present in the endoplasmic reticulum (ER). This protein is primarily involved in the regulation of intracellular Ca(2+) homeostasis and Ca(2+) storage in the ER. CRT also plays a significant role in autoimmunity and cancer. This protein contains three distinct structural domains with specialized functions. Here, we are reporting a simple procedure for the purification of CRT from mammalian kidney. To isolate CRT, sheep kidney was crushed and kept for 12 h in the extraction buffer. The lysate was centrifuged, and supernatant was precipitated by ammonium sulphate. The precipitate of 90 % ammonium sulphate was extensively dialyzed and loaded on DEAE-Hi-Trap FF and Mono Q chromatography columns. The purity of CRT was confirmed by SDS-PAGE. Finally, the protein was identified by matrix-assisted laser desorption/ionization time of flight. The purified protein was further characterized for secondary structural elements using the far-UV circular dichroism measurements. Our purification procedure is fast and simple with high yield.

Transient transfection of the human HEK293-EBNA1 cell line using polyethyleneimine is widely adopted for recombinant protein production. Whereas high expression of many targets is achieved, purification yields of some highly expressed proteins remain low due to aggregation. We hypothesized that for these proteins the expression rates achieved at standard transfection conditions are too high, causing an overload of the protein folding machinery. Here we present plasmid titration as an efficient method to vary expression rates for the optimization of soluble protein expression. In plasmid titration a dilution series of expression vector mixed with dummy plasmid is transfected in small scale cultures. Application to GFP shows that plasmid titration achieves a wide range of expression levels while maintaining high transfection efficiencies even at 500-fold plasmid dilution. Application of plasmid titration to selected Nod-like receptors (NLRs), which at standard conditions are highly expressed but poorly soluble, delays the onset of NLR aggregation and improves cell viability and the buildup of biomass. The amount of soluble protein depends on the combination of dilution factor and harvest day in a protein specific manner. For NOD1 50-fold plasmid dilution increases the amount of soluble protein approximately 5-fold. Due to its association with chaperones at all dilution factors tested we were unable to purify NOD1 to homogeneity. For NLRC4, which did not associate with chaperones, 10-fold plasmid dilution increased the purification yield 2-fold. This improvement, obtained with minimal effort due to the simplicity of the method, shows that reducing total expression may increase soluble protein yield., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents.

SCaMC is an ATP-Mg/Pi carrier protein located at the mitochondrial inner membrane. SCaMC has an unusual N-terminal Ca(2+)-binding domain (NTD) in addition to its characteristic six-helix transmembrane bundle. The NTD of human SCaMC1 (residues 1-193) was expressed and purified in order to study its role in Ca(2+)-regulated ATP-Mg/Pi transport mediated by its transmembrane domain. While Ca(2+)-bound NTD could be crystallized, the apo state resisted extensive crystallization trials. Selenomethionine-labeled Ca(2+)-bound NTD crystals, which belonged to space group P6(2)22 with one molecule per asymmetric unit, diffracted X-rays to 2.9 Å resolution.

Soybean protein hydrolysates (SPHs) can bind calcium in order to form soluble peptide-calcium complexes. However, amino acid composition and structural characteristics of the calcium chelating SPHs are still unclear. This study separated SPHs with calcium and iron immobilized metal affinity chromatography (IMAC), and examined the effects of SPHs with different amino acid composition on calcium binding capacity. Three fractions (FFe-1, FFe-2 and FFe-3) isolated with IMAC-Fe(3+) were shown possessing increased Glu, Gln, Lys and Pro content from FFe-1 to FFe-3, and improved amount of bound calcium. Furthermore, the fractions adsorbed on IMAC-Ca(2+) (Fe(3+)) were separated and identified with reverse-phase (RP)-HPLC and MALDI-TOF MS/MS. The results showed that the sequence of peptides from FCa-2 and FFe-3 fractions was DEGEQPRPFPFP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Crayfish sarcoplasmic calcium-binding protein (SCP) was purified. The physicochemical and polymorphic characterisations were also analysed. SCP was purified by column chromatography to reveal a single band with molecular mass of 22 kDa and further confirmed by mass spectrometry. The results of physicochemical characterisation showed that SCP was stable in the processes of thermal or acid/alkali treatment, and could be digested by simulate gastrointestinal fluid. Importantly, the comparison of SCP polymorphism using sera from crustacean-allergic patients demonstrated SCP-II had a weaker IgE-binding activity. The isoelectric points of SCP subunits a, b and c were 4.6, 4.7, and 4.8, respectively, as determined by two-dimensional electrophoresis and IgE immunoblotting analysis showed that patients' sera reacted to three subunits of SCP. Finally, it can be concluded that SCP is a stable polymorphic allergen in crayfish, and all of its isotypes and subunits have allergenicity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Nucleus pulposus from the porcine intervertebral disc was separated chromatographically to discover substrates of microbial transglutaminase. Highly purified proteins were prepared, among them type II collagen, the major protein of the nucleus pulposus. Determination of substrates was performed by transglutaminase-mediated incorporation of biotinylated probes displaying several glutamine and lysine donor proteins. Type II collagen was only labeled if smaller nucleus pulposus proteins were present. One of the modulating proteins was serotransferrin, a lysine donor substrate of bacterial transglutaminase. An additional substrate was the carboxy-terminal propeptide of type II collagen, chondrocalcin. Chondrocalcin, a regulator of type II collagen fibrillogenesis, occurs abundantly in juvenile cartilage and nucleus pulposus. Accordingly, the protein may be regarded as an excellent additive for the preparation of injectable stem cells in nucleus pulposus-like matrices cross-linked by microbial transglutaminase.

The mammalian sperm acrosome reaction is a calcium-dependent exocytotic event characterized by extensive fusion between the plasma and the outer acrosomal membrane. The mechanisms by which elevation of cytosolic calcium initiates the membrane fusion process are not understood and the present study was undertaken to identify calcium-binding proteins in the acrosomal membrane (AM) of bovine spermatozoa. Sperm heads, purified from sonicated spermatozoa, were used to isolate an acrosomal membrane-enriched fraction on Percoll density gradients. Using SDS-PAGE and a (45)Ca(2+)-blot overlay assay, calcium-binding proteins of 64, 45, 43, and 39kDa were identified in the AM enriched fraction. Phase separation analysis with Triton X-114 identified the 64kDa polypeptide as an integral membrane protein. The 64kDa polypeptide was purified and utilized to prepare a polyclonal antiserum. Both light and electron microscopic immunocytochemistry demonstrated that the protein was distributed throughout all domains of the acrosomal membrane. These results identify a 64kDa calcium-binding integral membrane protein of the mammalian acrosome. Its potential function in calcium-dependent membrane fusion events of the acrosome reaction and in fertilization is discussed., (Copyright © 2013. Published by Elsevier Ltd.)

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m(3) for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens.

Phylogenetic analysis of known dockerins in Ruminococcus flavefaciens revealed a novel subtype, type-III, in the scaffoldin proteins, ScaA, ScaB, ScaC and ScaE. In this study, we explored the Ca²⁺-binding properties of the type-III dockerin from the ScaA scaffoldin (ScaADoc) using a battery of structural and biophysical approaches including circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. Despite the lack of a second canonical Ca²⁺-binding loop, the behaviour of ScaADoc is similar with respect to other dockerin protein modules in terms of its responsiveness to Ca²⁺ and affinity for the cohesin from the ScaB scaffoldin. Our results highlight the robustness of dockerin modules and how their Ca²⁺-binding properties can be exploited in the construction of designer cellulosomes., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit. In this protocol, we describe the purification and isotope labeling strategies for productive NMR studies of CIB1. The same isotope labeling strategies can be implemented to study numerous related regulatory calcium-binding proteins.

EF-hand Ca(2+)-binding motif, a structural component of the EF-hand protein, functions as a calcium sensor and/or buffer in the cytosol of the cell. However, in a few exceptional cases, the EF-hand proteins are secreted from cells and play crucial roles extracellularly. We have identified longistatin, an EF-hand Ca(2+)-binding protein, from the salivary glands of the tick, Haemaphysalis longicornis. Longistatin possesses an N-terminal sequence of unknown structure and two EF-hand motifs in the C-terminus, which conserve a calmodulin-like canonical structure. Longistatin shows distinct changes in its migration during electrophoresis through SDS-PAGE gel containing calcium or ethylenediaminetetraacetic acid (EDTA). Both recombinant and endogenous forms of longistatin can be stained with rutheninum red, demonstrating that longistatin is a Ca(2+)-binding protein.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)