Colorimetric Cotton Swab for Viral Protease Detection.

Reporting the activity of a specific viral protease remains an acute need for rapid point-of-care detection strategies that can distinguish active infection from a resolved infection. In this work, we present a simple colorimetric approach for reporting the activity of a specific viral protease through direct color conversion on a cotton swab, which has the potential to be extended to detect the corresponding virus. We use SARS-CoV-2 viral protease as a proof-of-concept model system. We use 4-aminomalachite green (4-AMG) as the base chromophore structure to design a CoV2-AMG reporter, which is selective toward the SARS-CoV-2 Mpro but does not produce any observable color change in the presence of other viral proteases. The color change is observable by the naked eye, as well as smartphone imaging, which affords a lower limit of detection. The simplicity and generalizability of the method could be instrumental in combating future viral outbreaks.

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence.

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP.

The ability for biologics to access intracellular targets hinges on the translocation of active, unmodified proteins. This is often achieved using nanoscale formulations, which enter cells through endocytosis. This uptake mechanism often limits the therapeutic potential of the biologics, as the propensity of the nanocarrier to escape the endosome becomes the key determinant. To appropriately evaluate and compare competing delivery systems of disparate compositions, it is therefore critical to assess endosomal escape efficiencies. Unfortunately, quantitative tools to assess endosomal escape are lacking, and standard approaches often lead to an erroneous interpretation of cytosolic localization. In this study we use a split-complementation endosomal escape (SEE) assay to evaluate levels of cytosolic caspase-3 following delivery by polymer nanogels and mesoporous silica nanoparticles. In particular, we use SEE as a means to enable the systematic investigation of the effect of polymer composition, polymer architecture (random vs block), hydrophobicity, and surface functionality. Although polymer structure had little influence on endosomal escape, nanogel functionalization with cationic and pH-sensitive peptides significantly enhanced endosomal escape levels and, further, significantly increased the amount of nanogel per endosome. This work serves as a guide for developing an optimal caspase-3 delivery system, as this caspase-3 variant can be easily substituted for a therapeutic caspase-3 cargo in any system that results in cytosolic accumulation and cargo release. In addition, these data provide a framework that can be readily applied to a wide variety of protein cargos to assess the independent contributions of both uptake and endosomal escape of a wide range of protein delivery vehicles.

Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis.

Caspases are cysteine-aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23-45, and core of the caspase-6 structure. We observed that substitutions of the tri-arginine patch Arg-42-Arg-44 or the R44K cancer-associated mutation in caspase-6 markedly alter its rates of protein substrate hydrolysis. Notably, turnover of protein substrates but not of short peptide substrates was affected by these exosite alterations, underscoring the importance of this region for protein substrate recruitment. Hydrogen-deuterium exchange MS-mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the NTD and the 240's region (containing residues 236-246), which serves as a general exosite for caspase-6-specific substrate recruitment. In summary, we have identified an exosite on caspase-6 that is critical for protein substrate recognition and turnover and therefore highly relevant for diseases such as cancer in which caspase-6-mediated apoptosis is often disrupted, and in neurodegeneration in which caspase-6 plays a central role.

Multiple proteolytic events in caspase-6 self-activation impact conformations of discrete structural regions.

Caspase-6 is critical to the neurodegenerative pathways of Alzheimer's, Huntington's, and Parkinson's diseases and has been identified as a potential molecular target for treatment of neurodegeneration. Thus, understanding the global and regional changes in dynamics and conformation provides insights into the unique properties of caspase-6 that may contribute to achieving control of its function. In this work, hydrogen/deuterium exchange MS (H/DX-MS) was used to map the local changes in the conformational flexibility of procaspase-6 at the discrete states that reflect the series of cleavage events that ultimately lead to the fully active, substrate-bound state. Intramolecular self-cleavage at Asp-193 evoked higher solvent exposure in the regions of the substrate-binding loops L1, L3, and L4 and in the 130s region, the intersubunit linker region, the 26-32 region as well as in the stabilized loop 2. Additional removal of the linker allowed caspase-6 to gain more flexibility in the 130s region and in the L2 region converting caspase-6 to a competent substrate-binding state. The prodomain region was found to be intrinsically disordered independent of the activation state of caspase-6; however, its complete removal resulted in the protection of the adjacent 26-32 region, suggesting that this region may play a regulatory role. The molecular details of caspase-6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutic approaches for neurodegenerative disorders.

A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates.

Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging. Here, we constructed an outer membrane protein G (OmpG) nanopore with a caspase substrate sequence DEVDG grafted into one of the OmpG loops. Cleavage of the substrate sequence in the nanopore by caspase-7 generated a characteristic signal in the current recording of the OmpG nanopore that allowed the determination of the activity of caspase-7 in Escherichia coli cell lysates. Our approach may provide a framework for the activity-based profiling of proteases that share highly similar substrate specificity spectrums.

Cysteine Disulfide Traps Reveal Distinct Conformational Ensembles in Dengue Virus NS2B-NS3 Protease.

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.

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.

Caspase-9 CARD : core domain interactions require a properly formed active site.

Caspase-9 is a critical factor in the initiation of apoptosis and as a result is tightly regulated by many mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work, we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Taken together, these findings provide mechanistic insights into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate.

Active site-adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9.

Caspase-9 (casp-9) is an initiator caspase and plays a central role in activating apoptotic cell death. Control of all caspases is tightly regulated by a series of phosphorylation events enacted by several different kinases. Caspase-9 is the most heavily phosphorylated of all caspases, with phosphorylation of at least 11 distinct residues in all three caspase-9 domains by nine kinases. Caspase-9 phosphorylation by the non-receptor tyrosine kinase c-Abl at Tyr-153 reportedly leads to caspase-9 activation. All other phosphorylation events on caspases have been shown to block proteolytic function by a number of mechanisms, so we sought to unravel the molecular mechanism of the putative caspase-9 activation by phosphorylation. Surprisingly, we observed no evidence for Tyr-153 phosphorylation of caspase-9 in vitro or in cells, suggesting that Tyr-153 is not phosphorylated by c-Abl. Instead, we identified a new site for c-Abl-mediated phosphorylation, Tyr-397. This residue is adjacent to the caspase-9 active site but, as a member of the second shell, not a residue that directly contacts substrate. Our results further indicate that Tyr-397 is the dominant site of c-Abl phosphorylation both in vitro and upon c-Abl activation in cells. Of note, phosphorylation at this site inhibits caspase-9 activity, and the bulk of the added phosphate moiety appeared to directly block substrate binding. c-Abl plays both proapoptotic and prosurvival roles, and our findings suggest that c-Abl's effects on caspase-9 activity promote the prosurvival mode.

Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding.

Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a ß-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.

Lipogels for Encapsulation of Hydrophilic Proteins and Hydrophobic Small Molecules.

Lipid-polymer hybrid materials have the potential to exhibit enhanced stability and loading capabilities in comparison to parent liposome or polymer materials. However, complexities lie in formulating and characterizing such complex nanomaterials. Here we describe a lipid-coated polymer gel (lipogel) formulated using a single-pot methodology, where self-assembling liposomes template a UV-curable polymer gel core. Using fluorescently labeled lipids, protein, and hydrophobic molecules, we characterized their formation, purification, stability, and encapsulation efficiency via common instrumentation methods such as dynamic light scattering (DLS), matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), UV-vis spectroscopy, fluorescence spectroscopy, and single-particle total internal reflection fluorescence (TIRF) microscopy. In addition, we confirmed that these dual-guest-loaded lipogels are stable in solution for several months. The simplicity of this complete aqueous formation and noncovalent dual-guest encapsulation holds potential as a tunable nanomaterial scaffold.

Tumor-Associated Mutations in Caspase-6 Negatively Impact Catalytic Efficiency.

Unregulated, particularly suppressed programmed cell death is one of the distinguishing features of many cancer cells. The cysteine protease caspase-6, one of the executioners of apoptotic cell death, plays a crucial role in regulation of apoptosis. Several somatic mutations in the CASP6 gene in tumor tissues have been reported. This work explores the effect of CASP6 tumor-associated mutations on the catalytic efficiency and structure of caspase-6. In general, these mutations showed decreased overall rates of catalytic turnover. Mutations within 8 Å of the substrate-binding pocket of caspase-6 were found to be the most catalytically deactivating. Notably, the R259H substitution decreased activity by 457-fold. This substitution disrupts the cation-π stacking interaction between Arg-259 and Trp-227, which is indispensable for proper assembly of the substrate-binding loops in caspase-6. Sequence conservation analysis at the homologous position across the caspase family suggests a role for this cation-π stacking in the catalytic function of caspases generally. These data suggest that caspase-6 deactivating mutations may contribute to multifactorial carcinogenic transformations.

Utilizing Inverse Emulsion Polymerization To Generate Responsive Nanogels for Cytosolic Protein Delivery.

Therapeutic biologics have various advantages over synthetic drugs in terms of selectivity, their catalytic nature, and, thus, therapeutic efficacy. These properties offer the potential for more effective treatments that may also overcome the undesirable side effects observed due to off-target toxicities of small molecule drugs. Unfortunately, systemic administration of biologics is challenging due to cellular penetration, renal clearance, and enzymatic degradation difficulties. A delivery vehicle that can overcome these challenges and deliver biologics to specific cellular populations has the potential for significant therapeutic impact. In this work, we describe a redox-responsive nanoparticle platform, which can encapsulate hydrophilic proteins and release them only in the presence of a reducing stimulus. We have formulated these nanoparticles using an inverse emulsion polymerization (IEP) methodology, yielding inverse nanoemulsions, or nanogels. We have demonstrated our ability to overcome the liabilities that contribute to activity loss by delivering a highly challenging cargo, functionally active caspase-3, a cysteine protease susceptible to oxidative and self-proteolytic insults, to the cytosol of HeLa cells by encapsulation inside a redox-responsive nanogel.

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.

Reactive Self-Assembly of Polymers and Proteins to Reversibly Silence a Killer Protein.

Conjugation of biologically active proteins to polymeric materials is of great interest in the treatment of cancer and other diseases of protein deficiency. The conjugation of such biomacromolecules is challenging both due to their hydrophilicity and propensity to denature under non-native conditions. We describe a novel reactive self-assembly approach to "wrap" a protein with polymers, simultaneously protecting its delicate folded state and silencing its enzymatic activity. This approach has been demonstrated using caspase-3, an apoptosis-inducing protein, as the first case study. The protein-polymer conjugation is designed to be reversed under the native conditions for caspase-3, that is, the reducing environment found in the cytosol. The current strategy allowed release and recovery of up to 86% of caspase activity and nanogel-caspase-3 conjugates induced 70-80% apoptotic cell death shortly thereafter. This approach is widely generalizable and should be applicable to the intracellular delivery of a wide range of therapeutic proteins for treatment of complex and genetic diseases.

A Non-Active-Site Inhibitor with Selectivity for Zika Virus NS2B-NS3 Protease.

Flavivirus infection usually results in fever accompanied by headache, arthralgia, and, in some cases, rash. Although the symptoms are mild, full recovery can take several months. Flaviviruses encode seven nonstructural proteins that represent potential drug targets for this viral family. Focusing on the Zika virus NS2B-NS3 protease, we uncovered a unique inhibitor, MH1, composed of aminothiazolopyridine and benzofuran moieties. MH1 inhibits ZVP with a biochemical IC50 of 440 nM and effectively blocks cleavage of ZVP substrates in cells. Surprisingly, MH1 inhibits the other flaviviral proteases at least 18-fold more weakly. This same phenomenon was observed in assays of the viral cytopathic effect, where only Zika virus showed sensitivity to MH1. This selectivity was unexpected since flaviviral proteases have high similarity in sequence and protein structure. MH1 binds at an allosteric site, as demonstrated by its ability to stabilize ZVP synergistically with an active site inhibitor. To understand its selectivity, we constructed a series of hybrid proteases composed of select segments of ZVP, which is sensitive to MH1, and dengue virus protease, which is essentially insensitive to MH1. Our results suggest that MH1 binds to the NS3 protease domain, disrupting its interaction with NS2B. These interactions are essential for substrate binding and cleavage. In particular, the unique dynamic properties of NS2B from Zika seem to be required for the function of MH1. Insights into the mechanism of MH1 function will aid us in developing non-active-site-directed, pan-flaviviral inhibitors, by highlighting the importance of evaluating and considering the dynamics of the NS2B regions.

A tunable, modular approach to fluorescent protease-activated reporters.

Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring.

Zinc-mediated allosteric inhibition of caspase-6.

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.