Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death.

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.

Targeting TRAIL death receptor 4 with trivalent DR4 Atrimer complexes.

TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G(2), showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL.

A common mechanism for mitotic inactivation of C2H2 zinc finger DNA-binding domains.

Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chromatin condensation and cell division. C2H2 zinc fingers define the largest transcription factor family in the human proteome. The linker separating finger motifs is highly conserved and resembles TGEKP in more than 5000 occurrences. However, the reason for this conservation is not fully understood. We demonstrate that all three linkers in the DNA-binding domain of Ikaros are phosphorylated during mitosis. Phosphomimetic substitutions abolished DNA-binding and pericentromeric localization. A linker within Sp1 was also phosphorylated, suggesting that linker phosphorylation provides a global mechanism for inactivation of the C2H2 family.

Widespread failure of hematolymphoid differentiation caused by a recessive niche-filling allele of the Ikaros transcription factor.

A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. Ikaros(Plastic) homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies.