288 Hardiman: Role of research in developing anthrax countermeasures lular proteins in AT translocation, but also because these cellular proteins represent at- tractive novel targets for possible therapeu- tic intervention. The classical paradigm in the field was that cellular receptors have a role only at the initial steps in AT trafficking. After PA is se- creted from B. anthracis, it can bind to ubiq- uitous cellular receptors ANTXR1 [31] or ANTXR2 [32]. PA binding to ANTXR1 or ANTXR2 renders it susceptible to prote- olytic cleavage by furin proteases [33], yielding a 63-kDa peptide (PA63) that is ca- pable of binding LF and EF substrates [34]. After substrate binding is initiated, the PA63 prepore [11] and bound substrate are endo- cytosed through a clathrin-dependent mech- anism [35,36]. Events downstream of these initial actions have been assumed to be largely independent of cellular cofactors. The traditional model posited that sub- strate denaturation and translocation were driven solely by only changes in pH [23] in the endosomal microenvironment or electric potential in artificial systems [37,38]. Once environmental changes partially denature substrates [39], unfolding and protein “ra- cheting” through the pore was thought to be self-sustaining. This is likely mediated through the Φ and α clamps, which may act to preserve the proton gradient driving translocation [25] and support an unfolded conformation by non-specific binding [40], respectively. Indeed, support for this was shown that in artificial lipid bilayers con- taining only phospholipids and assembled AT, translocation was observed with no fur- ther requirements for exogenous cellular proteins [25,37]. However, recent work has raised the possibility that although AT com- ponents are both necessary and sufficient for substrate translocation, cellular proteins may further optimize this process. Recently, cellular proteins have been implicated in both the early process of sub- strate unfolding and the late process of pro- tein refolding after a substrate has completed translocation. The N-terminal domain of LF (LFn) contains the PA-binding domain and is used as a model peptide for stimulating ef- ficient translocation [41,42]. Diptheria Toxin fragment A (DTA) is often used as a model non-native substrate for the PA pore. This catalytic domain contains ADP-ribosy- lation activity, which causes death in sus- ceptible cells. When LFn is fused to DTA (LFnDTA), translocation can be measured as a function of cell death resulting from ADP-ribosylation of cellular proteins [43]. Using this approach, Tamayo and colleagues investigated the role for cellular proteins that enhance the native unfoldase activity of the PA pore [44]. Using a trypsin protease pro- tection assay, they identified the eukaryotic chaperone BiP (GRP78) as acting to unfold LFnDTA. Importantly, siRNA-mediated knockdown of BiP prevented intoxication of J774 macrophages by LF and EF. Further, cellular extracts containing ß-Cop, a com- ponent of the eukaryotic coatomer protein I (CopI) complex, were found to enhance translocation of LFnDTA compared to basal levels of native LF [45]. As before, anti- body-mediated immunodepletion of ß-Cop from cellular extracts impaired this translo- cation. This study led to the characterization of another cellular cofactor involved in the dis- tal steps of substrate binding to the PA pore. After proteins complete translocation, they exit the pore in a linearized, unfolded state. Chaperone proteins must bind to the translo- cated substrates to mediate refolding into an active conformation. Dmochewitz and col- leagues recently reported that CypA and Hsp90, well-characterized cellular chaper- ones, are required for the restoration of LFnDTA ADP-Ribosylation activity [46]. Interestingly, downregulating these proteins with pharmacological inhibitors protected cells from intoxication. Thus, for cellular proteins that may en- hance both early and late steps during in- toxication, robust protection is observed when these proteins are downregulated through various mechanisms. Because of the preliminary experimental validation, these proteins may be attractive targets for future therapeutic interventions in human studies. Indeed, characterization of the anthrax toxin receptor has led to subsequent therapeutic interventions in combating angiogenesis