Evaluation of ToxTrak (Trademark) for Analysis of Protein Toxin Toxicity

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

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Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Applied and Environmental Microbiology ◽

10.1128/aem.02197-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6549-6559 ◽

Cited By ~ 6

Author(s):

Sabrina Wemhoff ◽

Roland Klassen ◽

Friedhelm Meinhardt

Keyword(s):

Kluyveromyces Lactis ◽

Killer Toxin ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Target Cells ◽

Directed Mutagenesis ◽

Content Type ◽

Protein Toxin ◽

Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

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Sequence of the M28 dsRNA: Preprotoxin Is Processed to an α/β Heterodimeric Protein Toxin

Virology ◽

10.1006/viro.1995.0007 ◽

1995 ◽

Vol 213 (2) ◽

pp. 341-351 ◽

Cited By ~ 39

Author(s):

MANFRED J. SCHMITT ◽

DONALD J. TIPPER

Keyword(s):

Protein Toxin

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Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1552-1561.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1552-1561

Author(s):

R Esteban ◽

R B Wickner

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Synthesis ◽

Major Coat Protein ◽

Double Stranded Rna ◽

Dsrna Molecule ◽

Protein Toxin ◽

New Type ◽

Viruslike Particles ◽

M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

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Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance

Molecular Pharmaceutics ◽

10.1021/acs.molpharmaceut.6b00987 ◽

2017 ◽

Vol 14 (5) ◽

pp. 1429-1438 ◽

Cited By ~ 14

Author(s):

Yingzhi Chen ◽

Meng Zhang ◽

Hongyue Jin ◽

Yisi Tang ◽

Aihua Wu ◽

...

Keyword(s):

Protein Toxin ◽

Pegylated Protein

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Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion

Blood ◽

10.1182/blood.v79.3.576.bloodjournal793576 ◽

1992 ◽

Vol 79 (3) ◽

pp. 576-585 ◽

Cited By ~ 1

Author(s):

ML Grossbard ◽

AS Freedman ◽

J Ritz ◽

F Coral ◽

VS Goldmacher ◽

...

Keyword(s):

Phase I ◽

B Cell ◽

Maximum Tolerated Dose ◽

In Vitro Cytotoxicity ◽

Bolus Infusion ◽

Protein Toxin ◽

Cytotoxicity Studies ◽

Ricin A

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin “blocked ricin.” The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.

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Whole-organism Transcriptomic Insight Into the Effects of Cytco, A Novel Nematotoxic Protein on the Pine Wood Nematode Bursaphelenchus Xylophilus

10.21203/rs.3.rs-148830/v1 ◽

2021 ◽

Author(s):

Ye Chen ◽

Xiang Zhou ◽

Kai Guo ◽

Sha-Ni Chen ◽

Xiu Su

Keyword(s):

Enrichment Analysis ◽

Transcriptome Profiling ◽

Bursaphelenchus Xylophilus ◽

Control Measures ◽

Effective Control ◽

Large Set ◽

Pine Wood Nematode ◽

Pine Wood ◽

Protein Toxin ◽

Insight Into

Abstract Background: The pine wood nematode Bursaphelenchus xylophilus is a worldwide destructive pest on Pinus trees and lacks effective control measures. Screening nematotoxic protein toxins has been conducted to develop new strategies for nematode control. Results: The present study provided initial insights into the responses of B. xylophilus exposed to a nematocidal cytolytic-like protein (CytCo) based on the transcriptome profiling. A large set of differentially expressed genes (1266 DEGs) were found related to nematode development, reproduction, metabolism, motion, and immune system. In response to the toxic protein, B. xylophilus upregulated DEGs encoding cuticle collagens, transporters, and cytochrome P450. In addition, many DEGs related to cell death, lipid metabolism, major sperm proteins, proteinases/peptidases, phosphatases, kinases, virulence factors, and transthyretin-like proteins were downregulated. And Gene Ontology enrichment analysis showed that CytCo treatment significantly affecting DEGs functioning in muscle contraction, lipid localization, MAPK cascade. The pathway richness of Kyoto Encyclopedia of Genes and Genomes showed that the DEGs were concentrated in lysosome and fatty acid degradation. The weight co-expression network analysis indicated that the hub genes affected by CytCo were associated with the nematode cuticular collagen. Conclusions: These results showed that the CytCo protein toxin could interference gene expression to produce multiple nematotoxic effects, providing initial insight into its control potential of pine wood nematode.

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Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919038117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 6086-6091 ◽

Cited By ~ 2

Author(s):

Xui-Fen Liu ◽

Junxia Wei ◽

Qi Zhou ◽

Bruce A. Molitoris ◽

Ruben Sandoval ◽

...

Keyword(s):

Half Life ◽

Proximal Tubular Cells ◽

Proximal Tubule Cells ◽

Tubular Cells ◽

Two Photon Microscopy ◽

Two Photon ◽

Protein Toxin ◽

Mouse Imaging ◽

Proximal Tubular ◽

Recombinant Immunotoxins

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration ofl-lysine. Our data suggests thatl-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

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