ABSTRACT
A vast number of genes of unknown function threaten to clog drug discovery pipelines. To develop therapeutic products from novel genomic targets, it will be necessary to correlate biology with gene sequence information. Industrialized mouse reverse genetics is being used to determine gene function in the context of mammalian physiology and to identify the best targets for drug development.
Subject(s)
Genome , Animals , Automation , Computational Biology , Drug Industry/methods , Gene Library , Genetic Techniques , Humans , Mice , Mice, Knockout , Phenotype , SoftwareABSTRACT
Apaf-1 is a mammalian homolog of CED-4 that regulates cell death by participating in a ternary complex with cytochrome c, and procaspase-9. In the case of CED-4, two splice variants exist. The smaller (CED-4S) is proapoptotic while the larger (CED-4L) contains a short in-frame insert and is anti-apoptotic. We cloned a murine variant of apaf-1, termed apaf-1L, which contains an eleven amino acid insert similar to a recently described human apaf-1L clone. apaf-1 and apaf-1L have similar distributions in adult and fetal tissues, although apaf-1L transcripts are more abundant. Apaf-1L, undergoes homomerization and heteromerization with Apaf-1 in yeast. Apaf-1L also binds to caspase-9 and a dominant-negative isoform of caspase-9. Unlike CED-4, neither Apaf-1 variant was lethal in yeast. However, both Apaf-1 and Apaf-1L elicit cell death when cotransfected with caspase-9 into 293 EBNA cells. Although Apaf-1L was more potent than Apaf-1, their biological properties were qualitatively similar.
Subject(s)
Caenorhabditis elegans Proteins , Protein Biosynthesis , Alternative Splicing , Animals , Apoptosis , Apoptotic Protease-Activating Factor 1 , Calcium-Binding Proteins/genetics , Caspase 9 , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Cloning, Molecular , Enzyme Precursors/metabolism , Helminth Proteins/genetics , Mice , Organ Specificity , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Proteins/genetics , Proteins/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Transfection , Two-Hybrid System TechniquesABSTRACT
In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell suicide program. Ced-4 facilitates the proteolytic activation of the caspase, Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing. To examine the interactions among these proteins they were expressed in Saccharomyces cerevisiae. Ced-3 and Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing activity. Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing, showing Ced-9 can independently antagonize the action of both proteins. Ced-3- but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors, CrmA and p35. Thus, besides its Ced-3- and Ced-9-dependent action in C. elegans, Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in yeast. Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9. In addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate binding motif eliminated both homomerization and cell killing. We suggest the caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer.
