scholarly journals Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

2007 ◽  
Vol 406 (2) ◽  
pp. 355-363 ◽  
Author(s):  
Rafael B. da Silveira ◽  
Ana C. M. Wille ◽  
Olga M. Chaim ◽  
Marcia H. Appel ◽  
Dilza T. Silva ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Venom Gland ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Venom Protein ◽  
Functional Studies ◽  
Brown Spider ◽  
Endothelial Cell Cultures ◽  
And Function

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.

scholarly journals Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line

2007 ◽  
Vol 195 (2) ◽  
pp. 213-228 ◽  
Author(s):  
Elisabeth Sambroni ◽  
Florence Le Gac ◽  
Bernard Breton ◽  
Jean-Jacques Lareyre
Keyword(s):  
Rainbow Trout ◽  
Expression Pattern ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Functional Characterization ◽  
Gene Expression Pattern ◽  
Cross Reactivity ◽  
Functional Studies ◽  
Cognate Receptor

In vertebrates, gonadotropins (GTHs) (FSH and LH) are two circulating pituitary glycoprotein hormones that play a major role in the regulation of gonadal functions, including gonadal cell proliferation/differentiation and steroidogenesis. In mammals, it is well known that their biological effects are mediated by highly specific membrane-bound receptors expressed preferentially on the somatic cells of the gonads. However, in fish, binding and functional studies have shown that cross-reactivity may occur in GTH receptors depending on the species. To understand the molecular mechanisms involved in GTH actions, functional characterization of trout GTH receptors and their gonadal gene expression pattern has been carried out. The present study describes the presence of two distinct GTH receptors in trout showing similarities with those of higher vertebrates but also differences in their structural determinants. In vitro functional studies demonstrate that rtLH specifically activates its cognate receptor (EC50 = 117 ng/ml), whereas purified rainbow trout FSH (rtFSH) activates FSHR but also LHR at supraphysiological doses (EC50 = 38 vs 598 ng/ml for FSHR and LHR respectively). The high doses of rtFSH required to activate LHR put into question the physiological relevance of this interaction. The use of heterologous chinook GTHs confirms the strong preference of each hormone for its cognate receptor. The gonadal expression pattern of the GTH receptor genes suggests that FSH may play an important role in regulating gonadal functions, not only at the early stages but also at the final stages of the male and female reproductive cycles, in addition to the LH pathway.

scholarly journals N-glycosylation critically regulates function of oxalate transporter SLC26A6

2016 ◽  
Vol 311 (6) ◽  
pp. C866-C873 ◽  
Author(s):  
R. Brent Thomson ◽  
Claire L. Thomson ◽  
Peter S. Aronson
Keyword(s):  
Plasma Membrane ◽  
Intestinal Secretion ◽  
Integral Membrane Proteins ◽  
Extracellular Loop ◽  
Transport Activity ◽  
Intact Cells ◽  
Functional Studies ◽  
And Function ◽  
Oxalate Transport

The brush border Cl−-oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.

scholarly journals Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy

2020 ◽  
Vol 47 (6) ◽  
pp. 800-809 ◽  
Author(s):  
Maxime Cadieux-Dion ◽  
Simone Meneghini ◽  
Chiara Villa ◽  
Dènahin Hinnoutondji Toffa ◽  
Ronny Wickstrom ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Missense Variant ◽  
Functional Expression ◽  
Functional Studies ◽  
Sorting Intolerant From Tolerant ◽  
First Case ◽  
And Function

Abstract:Purpose:Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy.Method:We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene.Results:We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation.Conclusions:This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

scholarly journals Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

2001 ◽  
Vol 21 (3) ◽  
pp. 731-742 ◽  
Author(s):  
Josef Kuhn ◽  
Ulrike Tengler ◽  
Stefan Binder
Keyword(s):  
Plant Mitochondria ◽  
Rna Stability ◽  
Processing System ◽  
Stem Loop ◽  
Functional Studies ◽  
In Vitro Processing ◽  
Rapid Amplification ◽  
And Function

ABSTRACT To determine the influence of posttranscriptional modifications on 3′ end processing and RNA stability in plant mitochondria, peaatp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3′ nonencoded nucleotides. A 3′ rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3′ ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3′ poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3′ nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3′ processed and stable transcripts.

scholarly journals Effective In Vitro Clearance ofPorphyromonas gingivalis by Fcα Receptor I (CD89) on Gingival Crevicular Neutrophils

2001 ◽  
Vol 69 (5) ◽  
pp. 2935-2942 ◽  
Author(s):  
Tetsuo Kobayashi ◽  
Kouji Yamamoto ◽  
Noriko Sugita ◽  
Annemiek B. van Spriel ◽  
Susumu Kaneko ◽  
...  
Keyword(s):  
Gingival Crevicular Fluid ◽  
Polymorphonuclear Neutrophils ◽  
Causative Pathogen ◽  
Effector Functions ◽  
Functional Studies ◽  
Candidate Target ◽  
Optimal Target ◽  
Crevicular Fluid ◽  
And Function

ABSTRACT Porphyromonas gingivalis has been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance of P. gingivalis from disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (FcγR and FcαR, respectively) expression and function by studying IgG- and IgA-mediated elimination of P. gingivalis. GCF PMN exhibited higher FcαRI and FcγRI levels and lower FcγRIIa and FcγRIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonized P. gingivalisthan PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that FcαRI represents a candidate target for FcR-directed immunotherapy for the clearance of P. gingivalis.

scholarly journals Localization and Functional Characterization of Pulmonary Bovine Odorant-Binding Protein

2010 ◽  
Vol 48 (6) ◽  
pp. 1054-1060 ◽  
Author(s):  
G. B. Mitchell ◽  
M. E. Clark ◽  
R. Lu ◽  
J. L. Caswell
Keyword(s):  
Inflammatory Mediators ◽  
Binding Protein ◽  
Pulmonary Inflammation ◽  
Functional Characterization ◽  
Odorant Binding Protein ◽  
Surface Epithelium ◽  
Functional Studies ◽  
Flight Mass Spectrometry ◽  
Odorant Binding

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Functional Characterization of a Venom Protein Calreticulin in the Ectoparasitoid Pachycrepoideus vindemiae

Insects ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 29 ◽  
Author(s):  
Lei Yang ◽  
Beibei Wang ◽  
Liming Qiu ◽  
Bin Wan ◽  
Yi Yang ◽  
...  
Keyword(s):  
Expression System ◽  
Functional Characterization ◽  
Venom Gland ◽  
Amino Acid Identity ◽  
Immunohistochemical Localization ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Venom Protein ◽  
Pupal Parasitoids ◽  
Venom Proteins

Venom proteins act in the immunological interactions between parasitoids and their host insects. The effect of venom proteins on host immunity is not fully understood in pupal parasitoids. We identified the functions of a venom protein, calreticulin (PvCRT), in the pupal ectoparasitoid Pachycrepoideus vindemiae. Here, we report that PvCRT features a signal peptide and two conserved “calreticulin” domains. Multiple sequence alignments show that PvCRT shares 83.54% amino acid identity with CRT from both Pteromalus puparum and Nasonia vitripennis, which infers a close relationship among these three species. Using qPCR analysis, we found a lower expression level of PvCRT (0.27-fold) in the venom apparatus compared to the corresponding carcass. Immunohistochemical localization revealed that PvCRT was ubiquitously expressed in venom gland. The expression of the PvCRT gene in Drosophila transgenic lines via the UAS/Gal4 binary expression system reduced the self-encapsulation phenotype of tu(1)Sz1 mutants. Additionally, studies on humoral immunity indicate that PvCRT does not affect the antimicrobial immune responses of the host. This work on an ectoparasitoid will increase our understanding of venom–mediated host-parasitoid interactions.

scholarly journals Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jose Irimia-Dominguez ◽  
Chen Sun ◽  
Kunpeng Li ◽  
Barry B. Muhoberac ◽  
Grace I. Hallinan ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Functional Characterization ◽  
Protein Solubility ◽  
Cell Types ◽  
Brain Iron ◽  
Iron Storage ◽  
Human Ferritin ◽  
And Function ◽  
Brain Iron Metabolism

AbstractThe role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin’s three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin’s iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.

scholarly journals Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins

2018 ◽  
Vol 19 (11) ◽  
pp. 3418 ◽  
Author(s):  
Henrik O’Brien ◽  
Shingo Kanemura ◽  
Masaki Okumura ◽  
Robert Baskin ◽  
Pradip Bandyopadhyay ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Venom Gland ◽  
Cone Snail ◽  
Disulfide Isomerase ◽  
Folding Rates ◽  
Cone Snails ◽  
And Function ◽  
Protein Disulfide ◽  
Insight Into

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

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