Polymicrobial Synergy Stimulates Porphyromonas Gingivalis Survival And Gingipain Expression In A Multispecies Subgingival Community

Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment - modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three P. gingivalis strains in nutrient-rich broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient-rich broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. In the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that the ability of the community to grow was largely due to Rgp gingipain. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex serum substrates. Whereas they are constitutively expressed by P. gingivalis in nutrient-rich broth, gingipain expression in the model periodontal pocket environment (serum) appears to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which can then promote growth of the whole community.

A cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy

As a potent lymphocyte activator, interleukin-2 (IL-2) is an FDA-approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, we address these issues by engineering an IL-2 prodrug (ProIL2). We mask the activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Lastly, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Taken together, our approach presents an effective tumor targeting therapy with reduced toxicity.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

Impact of DJ-1 and Helix 8 on the Proteome and Degradome of Neuron-Like Cells

DJ-1 is an abundant and ubiquitous component of cellular proteomes. DJ-1 supposedly exerts a wide variety of molecular functions, ranging from enzymatic activities as a deglycase, protease, and esterase to chaperone functions. However, a consensus perspective on its molecular function in the cellular context has not yet been reached. Structurally, the C-terminal helix 8 of DJ-1 has been proposed to constitute a propeptide whose proteolytic removal transforms a DJ-1 zymogen to an active hydrolase with potential proteolytic activity. To better understand the cell-contextual functionality of DJ-1 and the role of helix 8, we employed post-mitotically differentiated, neuron-like SH-SY5Y neuroblastoma cells with stable over-expression of full length DJ-1 or DJ-1 lacking helix 8 (ΔH8), either with a native catalytically active site (C106) or an inactive site (C106A active site mutation). Global proteome comparison of cells over-expressing DJ-1 ΔH8 with native or mutated active site cysteine indicated a strong impact on mitochondrial biology. N-terminomic profiling however did not highlight direct protease substrate candidates for DJ-1 ΔH8, but linked DJ-1 to elevated levels of activated lysosomal proteases, albeit presumably in an indirect manner. Finally, we show that DJ-1 ΔH8 loses the deglycation activity of full length DJ-1. Our study further establishes DJ-1 as deglycation enzyme. Helix 8 is essential for the deglycation activity but dispensable for the impact on lysosomal and mitochondrial biology; further illustrating the pleiotropic nature of DJ-1.

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

The extracellular matrix (ECM) undergoes an orchestrated transition from embryonic to mature ECM that is essential for postnatal life, yet the developmental transition mechanisms for ECM components and macromolecular complexes are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes with important structural and regulatory roles. In mice, Fbn1 and Fbn2 mRNAs, which encode the major microfibrillar components, are strongly expressed during embryogenesis. Fbn2 mRNA levels rapidly decline postnatally, consistent with fibrillin-1 being the major component of adult tissue microfibrils. Here, by combining transgenic and N-terminomics strategies with in vitro analysis of microfibril assembly and intermolecular interactions, we identify cooperative proteolysis of fibrillin-2 by the secreted metalloproteases ADAMTS6 and ADAMTS10 as a mechanism contributing to postnatal fibrillin-1 dominance. The primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by demonstrating a dramatic reversal of skeletal defects in Adamts6−/− embryos by Fbn2 haploinsufficiency.

A novel theranostic activity-based probe targeting kallikrein 7 for diagnosis and treatment of skin diseases

We applied a new in silico approach for fishing protease-substrate motifs to design a kallirein 7 (KLK7)-specific phosphonate activity-based probe (ABP) to quantify the active KLK7 in situ. Epidermal application...

Development of a Structure-based Computational Simulation to Optimize the Blocking Efficacy of Pro-antibodies

Background: The on-target toxicity of monoclonal antibodies (Abs) is mainly due to the fact that Abs cannot distinguish target antigens (Ags) expressed in disease regions from those in normal tissues during systemic administration. In order to overcome this issue, we “copied” an autologous Ab hinge as an “Ab lock” and “pasted” it on the binding site of the Ab by connecting a protease substrate and linker in between to generate a pro-Ab, which can be specifically activated in the disease region to enhance Ab selectivity and reduce side effects. Previously, we reported that 70% of pro-Abs can achieve more than 100-fold blocking ability compared to the parental Abs. However, 30% of pro-Abs do not have such efficient blocking ability. This is because the same Ab lock linker cannot be applied to every Ab due to the differences in the complementarity-determining region (CDR) loops. Here we designed a method which uses structure-based computational simulation (MSCS) to optimize the blocking ability of the Ab lock for all Ab drugs. MSCS can precisely adjust the amino acid composition of the linker between the Ab lock and Ab drug with the assistance of molecular simulation.Results: We selected αPD-1, αIL-1β, αCTLA-4 and αTNFα Ab as models and attached the Ab lock with various linkers (L1 to L7) to form pro-Abs by MSCS, respectively. The resulting cover rates of the Ab lock with various linkers compared to the Ab drug were in the range 28.33%-42.33%. The recombinant pro-Abs were generated by MSCS prediction in order to verify the application of molecular simulation for pro-Ab development. The binding kinetics effective concentration (EC-50) for αPD-1 (200-250-fold), αIL-1β (152-186-fold), αCTLA-4 (68-150-fold) and αTNFα Ab (20-123-fold) were presented as the blocking ability of pro-Ab compared to the Ab drug. Further, there was a positive correlation between cover rate and blocking ability of all pro-Ab candidates. Conclusions: The results suggested that MSCS was able to predict the Ab lock linker most suitable for application to αPD-1, αIL-1β, αCTLA-4 and αTNFα Ab to form pro-Abs efficiently. The success of MSCS in optimizing the pro-Ab can aid the development of next-generation pro-Ab drugs to significantly improve Ab-based therapies and thus patients’ quality of life.

An automated protocol for modelling peptide substrates to proteases

Background Proteases are key drivers in many biological processes, in part due to their specificity towards their substrates. However, depending on the family and molecular function, they can also display substrate promiscuity which can also be essential. Databases compiling specificity matrices derived from experimental assays have provided valuable insights into protease substrate recognition. Despite this, there are still gaps in our knowledge of the structural determinants. Here, we compile a set of protease crystal structures with bound peptide-like ligands to create a protocol for modelling substrates bound to protease structures, and for studying observables associated to the binding recognition. Results As an application, we modelled a subset of protease–peptide complexes for which experimental cleavage data are available to compare with informational entropies obtained from protease–specificity matrices. The modelled complexes were subjected to conformational sampling using the Backrub method in Rosetta, and multiple observables from the simulations were calculated and compared per peptide position. We found that some of the calculated structural observables, such as the relative accessible surface area and the interaction energy, can help characterize a protease’s substrate recognition, giving insights for the potential prediction of novel substrates by combining additional approaches. Conclusion Overall, our approach provides a repository of protease structures with annotated data, and an open source computational protocol to reproduce the modelling and dynamic analysis of the protease–peptide complexes.

Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity

The Ananascomosus stem extract is a complex mixture containing various cysteine ​​proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 μM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.