ABSTRACT
The interleukin-1beta-converting enzyme-like protease precursor, pro-caspase-1, has an N-terminal prodomain that is removed during cleavage activation of the protease. Here we show that tumor necrosis factor treatment of HeLa cells induced apoptosis without detectable proteolytic activation of caspase-1 in the cytosol. Instead, tumor necrosis factor induced the translocation of pro-caspase-1 to the nucleus where it was proteolytically activated, releasing the intact prodomain. We identified a nuclear localization signal in the prodomain, which was required for translocation of both pro-caspase-1 as well as its prodomain to the nucleus. Surprisingly, transfected MCF-7 carcinoma or embryonic kidney 293T cells expressing the prodomain alone underwent apoptosis. These results show that death signal-induced nuclear targeting is a novel activity of a caspase prodomain and indicate that caspase-1 and its prodomain may have hitherto unsuspected nuclear functions in apoptosis.
Subject(s)
Caspases , Cell Nucleus/enzymology , Cysteine Endopeptidases/metabolism , Enzyme Precursors/metabolism , Amino Acid Sequence , Apoptosis , Breast Neoplasms , Caspase 1 , Cell Line , Cytosol/enzymology , Enzyme Activation , Epitopes/analysis , Epitopes/chemistry , Female , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Protein Processing, Post-Translational , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
A new gene encoding a 35.8-kDa mosquitocidal toxin (Mtx3; 326 amino acids) was isolated from Bacillus sphaericus SSII-1 DNA. Mtx3 is a new type of mosquitocidal toxin with homology to the Mtx2 mosquitocidal toxin of B. sphaericus SSII-1, the epsilon-toxin of Clostridium perfringens, and the cytotoxin of Pseudomonas aeruginosa. The mtx3 gene is highly conserved and widely distributed in both high- and low-toxicity mosquito larvicidal strains of B. sphaericus.
Subject(s)
Bacillus/genetics , Bacterial Toxins/genetics , Genes, Bacterial , Amino Acid Sequence , Bacterial Toxins/chemistry , Base Sequence , Clostridium perfringens/genetics , Conserved Sequence , DNA, Bacterial/genetics , Escherichia coli/genetics , Glutathione Transferase/genetics , Molecular Sequence Data , Molecular Weight , Pseudomonas aeruginosa/genetics , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
Five different mosquitocidal toxin (mtx2) gene homologs have been cloned from eight Bacillus sphaericus strains. Pairwise comparisons of the predicted amino acid sequences show between four and eight substitutions compared with the prototype Mtx2 from B. sphaericus strain SSII-1. Mtx2 from strain SSII-1 was approximately 7-fold more toxic to Culex mosquito larvae than the Mtx2 homolog from B. sphaericus strain 31-2. Conversely, Mtx2 from strain 31-2 was approximately 100-fold more toxic to Aedes mosquito larvae than Mtx2 from strain SSII-1. Lys224 in Mtx2 was found to be the most important amino acid for toxicity to Culex larvae, and substitution of Lys224 with threonine abolished the toxicity of Mtx2 from strain SSII-1 to these larvae. In complete contrast, Thr224 was found to be crucial for the toxicity of Mtx2 from strain 31-2 to Aedes larvae, and substitution of Thr224 with lysine caused a approximately 100-fold drop in toxicity to these larvae. Thus, amino acid 224 in the Mtx2 family of mosquitocidal toxins is an unusual and important determinant of mosquito larvicidal activity and host range.
