Jeanne A. Hardy

Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules.

Intracellular protein delivery is an important tool for both therapeutic and fundamental applications. Effective protein delivery faces two major challenges: efficient cellular uptake and avoiding endosomal sequestration. We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSCs). These NPSCs are formed and stabilized through supramolecular interactions between the nanoparticle, the protein cargo, and the fatty acid capsule interior. The NPSCs are ~130 nm in diameter and feature low toxicity and excellent stability in serum. The effectiveness of these NPSCs as therapeutic protein carriers was demonstrated through the delivery of fully functional caspase-3 to HeLa cells with concomitant apoptosis. Analogous delivery of green fluorescent protein (GFP) confirmed cytosolic delivery as well as intracellular targeting of the delivered protein, demonstrating the utility of the system for both therapeutic and imaging applications.

Mechanism of zinc-mediated inhibition of caspase-9

Zinc‐mediated inhibition is implicated in global caspase regulation, with relief of zinc‐mediated inhibition central to both small‐molecule and natively induced caspase activation. As an initiator, caspase‐9 regulates the upstream stages of the apoptotic caspase cascade, making it a critical control point. Here we identify two distinct zinc‐binding sites on caspase‐9. The first site, composed of H237, C239, and C287, includes the active site dyad and is primarily responsible for zinc‐mediated inhibition. The second binding site at C272 is distal from the active site. Given the amino‐acid conservation in both regions, these sites appear to be present across the caspase family underscoring the importance of zinc‐mediated regulation of this class of enzymes.

Zinc-mediated Allosteric Inhibition of Caspase-6

Background: Caspase-6 is a critical factor in neurodegeneration, which is regulated by zinc binding. Results: Caspase-6 is inhibited by zinc and binds one zinc/monomer at an exosite distal from the active site. Conclusion: Zinc allosterically inhibits caspase-6 by locking it into a naturally occurring, inactive, and extended helical conformation. Significance: The allosteric inhibition observed in the presence of zinc may aid in the development of allosteric caspase-6 drugs. Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation.

Substrate-induced conformational changes occur in all cleaved forms of caspase-6

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntingtons and Alzheimers diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60s and 130s helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

L2' loop is critical for caspase-7 active site formation.

The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2′) from the other half of the dimer to bind substrate. In an inactive form, the two L2′ loops form a cross‐dimer hydrogen‐bond network over the dimer interface. Although the L2′ loop has been implicated as playing a central role in the formation of the active‐site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2′ loop to catalytic function and enzyme stability. In wild‐type and all mutants, active‐site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand‐free conformation, but is as stable as wild type when bound to substrate, indicating that caspase‐7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2′ loop are shown to be essential for substrate‐binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side‐chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate‐binding are sufficiently strong to induce the active conformation. Thus, although the L2′ loop makes no direct contacts with substrate, it is essential for buttressing the substrate‐binding groove and is central to native catalytic efficiency.

Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2′ loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive ∼6000-fold reduction in catalytic efficiency. Mutations in the Arg-187–Tyr-223 couple have a far less dramatic effect (3–20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.

Allosteric inhibition of the NS2B-NS3 protease from dengue virus.

Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.

Co-delivery of protein and small molecule therapeutics using nanoparticle-stabilized nanocapsules.

Combination therapy employing proteins and small molecules provides access to synergistic treatment strategies. Co-delivery of these two payloads is challenging due to the divergent physicochemical properties of small molecule and protein cargos. Nanoparticle-stabilized nanocapsules (NPSCs) are promising for combination treatment strategies since they have the potential to deliver small molecule drugs and proteins simultaneously into the cytosol. In this study, we loaded paclitaxel into the hydrophobic core of the NPSC and self-assembled caspase-3 and nanoparticles on the capsule surface. The resulting combination NPSCs showed higher cytotoxicity than either of the single agent NPSCs, with synergistic action established using combination index values.

Using rigidity analysis to probe mutation-induced structural changes in proteins.

Predicting how a single amino acid substitution affects the stability of a protein structure is a fundamental task in macromolecular modeling. It has relevance to drug design and understanding of disease-causing protein variants. We present KINARI-Mutagen, a web server for performing in silico mutation experiments on protein structures from the Protein Data Bank. Our rigidity-based approach permits fast evaluation of the effects of mutations that may not be easy to perform in vitro, because it is not always possible to express a protein with a specific amino acid substitution. In two case studies we use KINARI-Mutagen to identify exposed residues that are known to be conserved, and we show that our prediction in the change in a proteins stability due to a mutation of an amino acid to glycine can be correlated against experimentally derived stability data. KINARI-Mutagen is available at http://kinari.cs.umass.edu.