GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death

The Mechanism of Inhibition of Antibody-Based Inhibitors of the Serine Protease MT-SP1

Due to the conserved nature of protease active sites, it has been difficult to develop protease inhibitors that are both potent and specific. The vast majority of both small molecule and naturally-occuring protein protease inhibitors target the active site, and while potent, are often promiscuous. With the goal of developing novel classes of protease inhibitors that would be both potent and specific, a phage-displayed antibody library was panned against the cancer-associated protease membrane-type serine protease 1 (MT-SP1). Two inhibitors were discovered from the screen, and their mechanism of inhibition was further studied to understand the basis of their potency and specificity. A number of kinetic and biochemical experiments revealed that while the two inhibitors, E2 and S4, bound to the enzyme with different binding mechanisms, they were both competitive inhibitors of both small-molecule and macromolecular MT-SP1 substrates. Mutational analysis by alanine scanning of the surface loops surrounding the protease active site revealed that the antibodies made numerous contacts with these loops. The surface loops are sites of natural diversity among closely related serine proteases, and created the epitope that allowed for antibody specificity. Taken together, these results suggest that these antibody inhibitors gain a measure of potency by binding in the protease active site, and additional potency and specificity through interactions with the protease surface loops. These strategies could be adapted in efforts to develop specific inhibitors against other proteases. A structure of the Fab construct of E2 in complex with the protease domain of MT-SP1 was determined by x-ray crystallography. The structure revealed that the inhibitor has a novel mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding a very large H3 loop in the active site in a catalytically non-competent manner. The results here illustrate that antibodies can be effective inhibitors because they can bind to features that traditional protease inhibitors do not. These lessons can be used to guide the development of molecules that can specifically inhibit any protease

Selective MALT1 paracaspase inhibition does not block TNF-a production downstream of TLR4 in myeloid cells

No abstract (Letter To The Editor

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition.

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 A resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold

Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation.

The NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly.To explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts.PKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly.Although PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway

Fragment-based protein-protein interaction antagonists of a viral dimeric protease

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose response determination was performed as a confirmation screen and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed via NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogs. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces