Ticks rely on salivary immunomodulators to sustain prolonged blood feeding and transmit pathogens, yet the protein effectors that mediate these processes remain incompletely defined. Here, we characterize three divergent cysteine protease inhibitors—Amacstatins—from the Gulf Coast tick Amblyomma maculatum and delineate their roles in feeding success and Rickettsia parkeri transmission. Integrated transcriptomic, structural, and biochemical analyses reveal that Amacstatin-1, -2, and -3 possess distinct reactive centers, inhibitory selectivity, and tissue-specific expression patterns that are dynamically regulated by blood feeding and rickettsial infection. Silencing all three Amacstatins via RNA interference significantly impaired tick feeding, reducing engorgement, fecundity, and R. parkeri burden in both midgut and salivary glands. Functional assays demonstrated that Amacstatins potently suppress host inflammatory responses, including pro-inflammatory cytokine production, nitric oxide release, and leukocyte recruitment. Together, these findings identify Amacstatins as critical immunomodulatory effectors that promote tick feeding and rickettsial transmission. This work provides a protein-level mechanistic insight into the A. maculatum–R. parkeri interaction and highlights Amacstatins as promising molecular targets for anti-tick and anti-rickettsial strategies.

Allergenic mites are a major source of indoor allergens and are linked to the increasing prevalence of allergic diseases, posing a growing global health concern. Protease allergens released by mites play a key role in the development of hypersensitivity by disrupting the epithelial barrier and inducing immune responses through their proteolytic activity. In this study, we show that cysteine proteases significantly contribute to the overall proteolytic capacity of allergenic mites, particularly in house dust mites, and are involved in the degradation of human extracellular matrix proteins. Using a functional proteomics approach, combining specific substrates, inhibitors, and activity-based imaging probes, we profiled the component proteases in the species Dermatophagoides farinae and Acarus siro. This analysis identified two novel types of abundant, active cysteine proteases related to cathepsins: cathepsin B from D. farinae (DfCB) and cathepsin C from A. siro (AsCC), which are excreted into the environment via mite feces. Both cathepsins are clearly distinct from canonical cysteine proteases belonging to mite group 1 allergens, as demonstrated by comprehensive biochemical and functional characterizations, together with sequence and phylogenetic analyses. We also validated selective substrates for the detection of DfCB and AsCC in complex proteomes. Our findings suggest that DfCB and AsCC function as digestive enzymes in mites, and that their potent proteolytic activity, retained after excretion, may contribute to interactions with human tissues involved in allergic sensitization.

Ticks are important ectoparasites and vectors of a variety of pathogens in both animals and humans, and their increasing global distribution poses a growing health risk. Unlike other blood-feeding vectors, ticks feed for an extended period at each life stage and rely exclusively on blood for development and reproduction. Blood digestion in ticks is mediated by a complex multienzyme network within the endolysosomal system of the midgut (MG) epithelial cells. Previous studies have focused largely on protein digestion during the slow feeding phase. However, the processing of the blood meal after the mating-induced rapid engorgement ("big sip") remains unclear, although the rapid turnover of proteins from host blood proteins into yolk proteins in fully fed females is a crucial step for tick reproduction. In this study, we performed a label-free quantitative proteomic analysis of MG tissue extracts and MG contents of the hard tick Ixodes ricinus to characterize proteases and protease inhibitors expressed during selected timepoints of female feeding and off-host digestion. In addition, we analyzed the distribution of digestive enzymes by activity profiling in MG extracts and contents with specific diagnostic substrates. Our results show that the multienzyme network, mainly based on aspartic acid and cysteine cathepsins and complemented by specific types of serine proteases and metalloproteases, is involved in the intracellular and probably also in the luminal digestion of blood meal proteins in fully engorged female ticks. We also detected different types of protease inhibitors and proposed their regulatory role in controlling both endogenous (tick-derived) and host protease activities in the MG tissue and luminal contents storing ingested blood. These results provide comprehensive insights into the physiology of the tick MG and offer new opportunities for the development of future control strategies against ticks and tick-borne diseases.

Cysteine and serine proteases cleave peptides through covalent catalysis by generating a transient adduct with the N-terminal part of the substrate after releasing its C-terminal part. We demonstrate the unique redirection of this event leading to strong enzyme inactivation. For targeting human cathepsin B, a cysteine protease of significant therapeutic importance, we designed tailored peptidomimetics with a variety of dipeptide fragments directed toward the occluding loop and equipped with numerous N-terminal carbamate warheads. The carbamate deprotonation catalyzed by the active site thiolate initiates the redirected cleavage. The C-terminal part of the inhibitors remains covalently attached to the protease. Hydrolysis of such carbamoyl-enzyme complexes is catalytically unsupported rendering inhibition irreversible. This novel mechanism of action comprises a significant extension of the covalent drug space.

Trematodes, a class of parasitic flatworms, are responsible for a variety of devastating diseases in humans and animals, with schistosomiasis and fascioliasis being prominent examples. Trematode proteolytic systems involved in the host-parasite interaction have emerged as key contributors to the success of trematodes in establishing and maintaining infections. This review concentrates on diverse proteases and protease inhibitors employed by trematodes and provides an update on recent advances in their molecular-level characterization, with a focus on function, structure, and therapeutic target potential.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1 ' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure–function characterization of IrThy—the first investigated thyropin of parasite origin—and suggests its potential role in host–parasite interactions at the tick bite site.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease in-hibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high -resolution crystal structure of FhCyLS-2 showed a distinct di-sulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, sug-gesting a dual role in the regulation of exogenous and endog-enous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Aim:Comprehensive, global information on species' occurrences is an essential biodiversity variable and central to a range of applications in ecology, evolution, biogeography and conservation. Expert range maps often represent a species' only available distributional information and play an increasing role in conservation assessments and macroecology. We provide global range maps for the native ranges of all extant mammal species harmonised to the taxonomy of the Mammal Diversity Database (MDD) mobilised from two sources, the Handbook of the Mammals of the World (HMW) and the Illustrated Checklist of the Mammals of the World (CMW). Location:Global. Taxon:All extant mammal species. Methods:Range maps were digitally interpreted, georeferenced, error-checked and subsequently taxonomically aligned between the HMW (6253 species), the CMW (6431 species) and the MDD taxonomies (6362 species). Results:Range maps can be evaluated and visualised in an online map browser at Map of Life (mol.org) and accessed for individual or batch download for non-commercial use. Main conclusion:Expert maps of species' global distributions are limited in their spatial detail and temporal specificity, but form a useful basis for broad-scale characterizations and model-based integration with other data. We provide georeferenced range maps for the native ranges of all extant mammal species as shapefiles, with species-level metadata and source information packaged together in geodatabase format. Across the three taxonomic sources our maps entail, there are 1784 taxonomic name differences compared to the maps currently available on the IUCN Red List website. The expert maps provided here are harmonised to the MDD taxonomic authority and linked to a community of online tools that will enable transparent future updates and version control.