Neutralization of cholera toxin by Rosaceae family plant extracts.

BACKGROUND: Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin). METHODS: Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts. RESULTS: The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process. CONCLUSIONS: The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

Transcriptional repressor DREAM regulates trigeminal noxious perception.

Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain-derived neurotrophic factor in trigeminal ganglia. Genome-wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception. This article is part of the special article series "Pain".

Bacterial type AB5 enterotoxins--structure, function and mechanism of action.

One of the main factors causing bacterial diarrhea are AB5 enterotoxins. This group is divided into four families: pertussis toxin, cholera toxin, shiga toxin and subtilase cytotoxin. In this review we will describe the activity, structure and function of the cholera and shiga toxin families. The AB5 enterotoxins contain a catalytic subunit A and pentameric subunit B, which binds to the cell surface within lipid rafts. The cholera toxin family cause the constitutive activation of Gsa protein, which results in cAMP production, an opening of the chloride channels and releases chloride ions into the lumen of the small intestine. In contrast, the shiga toxin family has a cytotoxic effect on epithelial cells. It can inhibit protein synthesis leading to cell death. Although AB5 has a toxic activity, the B5 subunits have a significant potential as a transporter for proteins with anticancer activity and as a tool for the visualization of lipid rafts and cancer cells.

Use of plant extracts to control and treat AB5 enterotoxin-related diarrhea.

Plants contain a broad spectrum of small molecules with potential antimicrobial properties. Here, we review the antimicrobial activities of plant extracts against enterotoxic bacteria encoding AB5 toxins, including Vibrio cholerae, Shigella dysenteriae and enterotoxic Escherichia coli strains. Several plant extracts have strong antimicrobial effects and the potential to boost Oral Rehydration Therapy, which is the first line of treatment for acute diarrhea.

Sumoylation regulates nuclear localization of repressor DREAM.

DREAM is a Ca(2+)-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

S100A proteins in propagation of a calcium signal in norm and pathology.

Calcium ions are essential factors controlling the balance between cell survival, growth, differentiation and metabolism. Ca2+ acts as a global second messenger involved in the regulation of all aspects of cell function. Fluctuations in the intra- and extracellular Ca2+ concentration [Ca2+] in response to different environmental stimuli drive most cellular functions. Therefore, sustenance of calcium homeostasis requires perfect organization in time and space that is achieved by calcium binding proteins (CaBPs). These proteins are involved in sensing and transforming calcium signals to downstream cellular responses. Growing number of evidence suggests than many human disorders, including cancer progression, are related to deregulation of cellular calcium homeostasis and defects in CaBPs functions. In this review we will focus on the roles of S100A proteins in intracellular and extracellular calcium signalling and homeostasis. The S100A subfamily is among the most distinctive of EF-hand CaBPs and are found exclusively in vertebrates. They are believed to have evolved to enable activation of specific biochemical pathways in parallel to the activity of Ca2+ sensors such as calmodulin and/or annexins. The importance of S100 proteins is underscored by their deregulated expression in neurodegenerative and inflammatory disorders, myopathies and cancer. In addition, S100 proteins serve as diagnostic markers in the clinic and are under constant investigation. Their roles and the roles of the S100A protein partners in normal and pathology will be also discussed.

Matrix vesicles isolated from mineralization-competent Saos-2 cells are selectively enriched with annexins and S100 proteins.

Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.

Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples.

Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers.

An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology.

De novo designed bioactive molecules, such as DNA, RNA and peptides, are utilized in increasingly diverse scientific, industrial and biomedical applications. Concatemerization of designed DNA, RNA and peptides may improve their stability, bioactivity and allow for gradual release of the bioactive molecule at the intended destination. In this context, we developed a new method enabling the formation of DNA concatemers for the production of artificial, repetitive genes, encoding concatemeric RNAs and proteins of any nucleotide and amino-acid sequence. The technology recruits the Type IIS SapI restriction endonuclease (REase) for assembling DNA fragments in an ordered head-to-tail-orientation. Alternatively, other commercially available SapI isoschizomers can be used: LguI and thermostable BspQI. Four series of DNA vectors dedicated to the expression of newly formed, concatemeric open reading frames (ORFs), were designed and constructed to meet the technology needs. • Vector-enzymatic DNA fragment amplification technology. • Construction of DNA concatemers many times longer than those available with the use of current de novo gene synthesis methods. • Biosynthesis of protein tandem repeats with programmable function never seen in nature.

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method.

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: (i) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, (ii) sequences of the designed primers, vectors and recombinant constructs, (iii) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

Day-night changes in downstream regulatory element antagonist modulator/potassium channel interacting protein activity contribute to circadian gene expression in pineal gland.

The molecular mechanisms controlling the oscillatory synthesis of melatonin in rat pineal gland involve the rhythmic expression of several genes including arylalkylamine N-acetyltransferase (AA-NAT), inducible cAMP early repressor (ICER), and Fos-related antigen-2 (fra-2). Here we show that the calcium sensors downstream regulatory element antagonist modulator/potassium channel interacting protein (DREAM/KChIP)-3 and KChIP-1, -2 and -4 bind to downstream regulatory element (DRE) sites located in the regulatory regions of these genes and repress basal and induced transcription from ICER, fra-2 or AA-NAT promoters. Importantly, we demonstrate that the endogenous binding activity to DRE sites shows day-night oscillations in rat pineal gland and retina but not in the cerebellum. The peak of DRE binding activity occurs during the day period of the circadian cycle, coinciding with the lowest levels of fra-2, ICER, and AA-NAT transcripts. We show that a rapid clearance of DRE binding activity during the entry in the night period is related to changes at the posttranscriptional level of DREAM/KChIP. The circadian pattern of DREAM/KChIP activity is maintained under constant darkness, indicating that an endogenous clock controls DREAM/KChIP function. Our data suggest involvement of the family of DREAM repressors in the regulation of rhythmically expressed genes engaged in circadian rhythms.

Characterization of calretinin I-II as an EF-hand, Ca2+, H+-sensing domain.

Calretinin, a neuronal protein with well-defined calcium-binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N-terminal (CR I-II), and C-terminal (CR III-VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium-bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The TNS data for the N- and C-terminal domains of CR are additive, providing further support for their independence within the full-length protein. Our work concentrated on CR I-II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I-II from a phenyl-Sepharose column was consistent with the TNS data. The pH-dependent structural changes were further localized to residues 13-28 and 44-51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N-terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.

Calretinin and calbindin D28k have different domain organizations.

The domain organization of calretinin (CR) was predicted to involve all six EF-hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed (1)H,(15)N HSQC NMR spectra of a (15)N-labeled calretinin fragment (CR III-VI, residues 100-271) in the presence of the unlabeled complimentary fragment (CR I-II, residues 1-100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of (15)N-labeled CR is similar to the overlaid spectra of individual (15)N-labeled CR fragments (CR I-II and CR III-VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1-60) with CR II-VI (61-271), and CR I-III (1-142) with CR IV-VI (145-271). We conclude that CR is formed from at least two independent domains consisting of CR I-II and CR III-VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase-3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF-hand proteins, and perhaps of other protein families.

Stimulators of mineralization limit the invasive phenotype of human osteosarcoma cells by a mechanism involving impaired invadopodia formation.

BACKGROUND: Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). RESULTS: In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. CONCLUSIONS: Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Temperature dependence of ligand-protein complex formation as reflected by saturation transfer difference NMR experiments.

We show that temperature is an important parameter for the sensitivity of saturation transfer difference (STD) spectroscopy. A decreased intensity of STD signals is observed for lactose binding to growth-regulatory galectin7 (p53-induced gene 1), as well as for nucleotide binding to annexin A6, when the temperature is increased from 281 to 298-310 K. Opposite temperature effects on STD intensity are observed for S-peptide binding to S-protein to reconstitute RNase S. However, the STD signals for tryptophan binding to downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM)are relatively unaffected between 281 and 298 K. The known kinetics of the binding of ATP by the uncoupling protein from brown adipose tissue mitochondria (UCP1) predicted an observable STD at 310 K, but rapid sample degradation limits the experiments to much lower temperatures. Temperature strongly influences the kinetics and affinity constant of various types of complex formation and in so doing influences the observed STD effects. Therefore, temperature can be exploited to facilitate the optimization of STD-based applications, and at the same time minimize the number of test samples. STD-based screening protocols to detect new target-specific compounds may yield a larger number of potential ligands if screened at various temperatures.

Transcriptional repressor DREAM regulates T-lymphocyte proliferation and cytokine gene expression.

Downstream Regulatory Element Antagonist Modulator (DREAM) is a Ca2+-dependent transcriptional repressor expressed in the brain, thyroid gland and thymus. Here, we analyzed the function of DREAM and the related protein KChIP-2 in the immune system using transgenic (tg) mice expressing a cross-dominant active mutant (EFmDREAM) for DREAM and KChIPs Ca2+-dependent transcriptional derepression. EFmDREAM tg mice showed reduced T-cell proliferation. Tg T cells exhibited decreased interleukin (IL)-2, -4 and interferon (IFN)gamma production after polyclonal activation and following antigen-specific response. Chromatin immunoprecipitation and transfection assays showed that DREAM binds to and represses transcription from these cytokine promoters. Importantly, specific transient knockdown of DREAM or KChIP-2 induced basal expression of IL-2 and IFNgamma in wild-type splenocytes. These data propose DREAM and KChIP-2 as Ca2+-dependent repressors of the immune response.

Concanavalin A-agarose removes mannan impurities from an extracellularly expressed Pichia pastoris recombinant protein.

Pichia pastoris secretes few native proteins. However, the more than 1 g l(-1)of extracellularly expressed mannan interfered with the purification of our extracellularly expressed, non-glycosylated recombinant protein. Concanavalin A-agarose removed more than 95% of the unwanted mannan as monitored by phenol reaction. A(13)C-based NMR assay confirmed this improvement. Concanavalin A-agarose can assist the purification of extracellular expressed, non-glycosylated proteins from yeasts.

Overexpressed yeast mitochondrial putative RNA helicase Mss116 partially restores proper mtRNA metabolism in strains lacking the Suv3 mtRNA helicase.

RNA helicase, encoded by the Saccharomyces cerevisiae nuclear gene SUV3, is a subunit of the mitochondrial (mt) degradosome: an enzyme complex that takes part in turnover of mtRNAs. Deletion of the SUV3 gene leads to a variety of disturbances in mtRNA metabolism and results in respiratory incompetence of yeast cells. Here we show that the nuclear gene MSS116, which codes for a mitochondrial putative RNA helicase necessary for splicing of several mt introns, can suppress the lack of the SUV3 gene. Overexpression of the Mss116 putative helicase from a multicopy plasmid present in the SUV3-deleted strains partially restores respiratory competence, brings the steady-state levels of COB and ATP6/8 mRNA back almost to normal and lowers the accumulation of 21S rRNA and ATP6/8 RNA precursors to the wild-type levels. To the best of our knowledge, this is the first reported case of a substitution of one RNA helicase by another, belonging to a different class of RNA helicases.