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#cell killing

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Immunological Applications

Immunotoxins kill target cells mainly by:

Mechanism by which protein toxins arrest protein synthesis culminates in rapid programmed cell death via mitochondrial pathway capable of killing quiescent cells. Diphtheria toxin fragment A catalyzes ADP-ribosylation of unusual post-translationally modified histidine diphthamide at position 699 of eukaryotic elongation factor 2 present exclusively on domain IV, using oxidized NAD+ as ADP-ribose donor releasing nicotinamide. Modified elongation factor cannot mediate translocation step of peptidyl-tRNA from A site to P site on 60S ribosomal subunit, halting elongation after single round. Ricin A chain functions as RNA N-glycosidase hydrolyzing N-glycosidic bond of adenine 4324 in 28S ribosomal RNA alpha sarcin-ricin loop critical for EF2 GTPase activation, depurinating single base rendering 60S subunit unable to bind elongation factors. Both events cause accumulation of stalled ribosomes triggering ribotoxic stress response via MAP3K ZAK activating JNK and p38, plus depletion of short-lived anti-apoptotic proteins Mcl-1 half-life thirty minutes, XIAP, c-FLIP required to restrain caspase cascade, tipping balance toward Bax Bak oligomerization forming mitochondrial outer membrane pores releasing cytochrome c, Smac DIABLO, formation of Apaf-1 apoptosome activating initiator caspase 9 then executioners caspase 3 and 7 cleaving PARP leading to chromatin condensation within hours even in nondividing cells resistant to mitotic inhibitors.

Ref: Collier Annu Rev Biochem 1975 diphtheria ADP-ribosyl EF2; Olsnes Pharmac Ther ribosome N-glycosidase ricin apoptosis.

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Immunological Applications

Immunotoxins kill target cells mainly by:

Protein toxins employed immunotoxin design kill primarily through catalytic inactivation protein synthesis machinery requiring only few molecules cytosol triggering irreversible apoptosis. Diphtheria toxin fragment A 21 kDa ADP-ribosyltransferase modifies diphthamide residue unique post-translationally modified histidine 699 eukaryotic elongation factor 2 synthesized seven enzymes DPH1-7 adding 3-amino-3-carboxypropyl trimethylation diphthine amidation transferring ADP-ribose oxidized NAD imidazole inhibiting translocation peptidyl tRNA A site P site elongation cycle 80S ribosome mediated GTP hydrolysis. Pseudomonas exotoxin A domain III similarly ADP-ribosylates same residue furin cleavage toxin endosome separating catalytic domain. Ricin A chain N-glycosidase hydrolyzes N-glycosidic bond depurinating adenine 4324 sarcin-ricin loop GAGA tetraloop 28S rRNA preventing binding elongation factors eEF1 aminoacyl tRNA eEF2 interfering factor-dependent GTPase. Arrest translation rapidly depletes short-lived antiapoptotic proteins MCL-1 half-life 2h XIAP continuous turnover releasing Bak Bax oligomerization forming pores outer mitochondrial membrane cytochrome c release activating apoptosome caspase-9 caspase-3 cascade executing death concentrations 10^-11 M explaining extraordinary potency exceeding conventional small-molecule chemotherapeutics lacking enzymatic amplification catalytic turnover.

Ref: Collier 1967 Diphtheria Toxin ADP-ribosylation EF2 Discovery; Pastan PE38 Protein Synthesis Inhibition Mechanism; Lodish Protein Synthesis eEF2 Function Chapter 7.

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