Kinase inhibitors are effective cancer therapies. Unfortunately, drug resistance emerges in response to kinase inhibition leading to loss of drug efficacy. In this issue of Cell Chemical Biology, Peh ...et al. (2018) demonstrate that caspase activators effectively delay onset of resistance to kinase inhibitors and are excellent co-therapeutics for a number of tumor types.
Kinase inhibitors are effective cancer therapies. Unfortunately, drug resistance emerges in response to kinase inhibition leading to loss of drug efficacy. In this issue of Cell Chemical Biology, Peh et al. (2018) demonstrate that caspase activators effectively delay onset of resistance to kinase inhibitors and are excellent co-therapeutics for a number of tumor types.
Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address ...this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.
Caspases, the cysteine proteases which facilitate the faithful execution of apoptosis, are tightly regulated by a number of mechanisms including phosphorylation. In response to cAMP, PKA ...phosphorylates caspase-9 at three sites preventing caspase-9 activation, and suppressing apoptosis progression. Phosphorylation of caspase-9 by PKA at the functionally relevant site Ser-183 acts as an upstream block of the apoptotic cascade, directly inactivating caspase-9 by a two-stage mechanism. First, Ser-183 phosphorylation prevents caspase-9 self-processing and directly blocks substrate binding. In addition, Ser-183 phosphorylation breaks the fundamental interactions within the caspase-9 core, promoting disassembly of the large and small subunits. This occurs despite Ser-183 being a surface residue distal from the interface between the large and small subunits. This phosphorylation-induced disassembly promotes the formation of ordered aggregates around 20 nm in diameter. Similar aggregates of caspase-9 have not been previously reported. This two-stage regulatory mechanism for caspase-9 has likewise not been reported previously but may be conserved across the caspases.
Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches ...that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH‐sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein‐modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self‐proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase‐3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild‐type caspase‐3. Only in the presence of cytosolic GFP strands 1–10 will the tagged caspase‐3 generate fluorescence to signal a non‐endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomally‐entrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp‐3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge–property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.
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.
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.
The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A ...number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an “open” (inactive) conformation and a “closed” (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the “closed” conformation dramatically increased activity, strongly suggesting that the “closed” conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an “unlinked” construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a “closed”, active conformation. In contrast, in a “linked” construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the “closed” and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the “unlinked” constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.
Caspases, the cysteine proteases that execute apoptosis, are tightly regulated via phosphorylation by a series of kinases. Although all apoptotic caspases work in concert to promote apoptosis, ...different kinases regulate individual caspases. Several sites of caspase-7 phosphorylation have been reported, but without knowing the molecular details, it has been impossible to exploit or control these complex interactions, which normally prevent unwanted proliferation. During dysregulation, PAK2 kinase plays an alternative anti-apoptotic role, phosphorylating caspase-7 and promoting unfettered cell growth and chemotherapeutic resistance. PAK2 phosphorylates caspase-7 at two sites, inhibiting activity using two different molecular mechanisms, before and during apoptosis. Phosphorylation of caspase-7 S30 allosterically obstructs its interaction with caspase-9, preventing intersubunit linker processing, slowing or preventing caspase-7 activation. S239 phosphorylation renders active caspase-7 incapable of binding substrate, blocking later events in apoptosis. Each of these mechanisms is novel, representing new opportunities for synergistic control of caspases and their counterpart kinases.
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•PAK2 phosphorylation of caspase-7 inhibits activity by two distinct mechanisms•Phosphorylation of caspase-7 at S30 slows zymogen activation by upstream caspases•S30 phosphorylation interferes with caspase-7:caspase-9 interaction•Crystal structure of S239E phosphomimetic suggests substrate binding is obstructed
PAK2 phosphorylates caspase-7 resulting in loss of activity, which is implicated in resistance to chemotherapeutic agents. Eron et al. decipher the molecular mechanism of phosphorylated caspase-7 inhibition at two phosphorylation sites. Phosphorylation at S30 slows initial activation while phosphorylation at S239 blocks substrate binding.
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.
The balance of pro-apoptotic and pro-survival proteins defines
a cell’s fate. These processes are controlled through an interdependent
and finely tuned protein network that enables survival or leads ...to
apoptotic cell death. The caspase family of proteases is central to
this apoptotic network, with initiator and executioner caspases, and
their interaction partners, regulating and executing apoptosis. In
this work, we interrogate and modulate this network by exogenously
introducing specific initiator or executioner caspase proteins. Each
caspase is exogenously introduced using redox-responsive polymeric
nanogels. Although caspase-3 might be expected to be the most effective
due to the centrality of its role in apoptosis and its heightened
catalytic efficiency relative to other family members, we observed
that caspase-7 and caspase-9 are the most effective at inducing apoptotic
cell death. By critically analyzing the introduced activity of the
delivered caspase, the pattern of substrate cleavage, as well as the
ability to activate endogenous caspases, we conclude that the efficacy
of each caspase correlated with the levels of pro-survival factors
that both directly and indirectly impact the introduced caspase. These
findings lay the groundwork for developing methods for exogenous introduction
of caspases as a therapeutic option that can be tuned to the apoptotic
balance in a proliferating cell.
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