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.

• Klíčová slova
• cathepsin, cysteine protease, parasite, protease inhibitor, protein structure, saliva, thyropin, tick,
• MeSH
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny metabolismus   MeSH
• klíště * metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• sliny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny MeSH

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 inhibitors 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 disulfide 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, suggesting a dual role in the regulation of exogenous and endogenous 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.

• Klíčová slova
• cystatin, cysteine cathepsin, helminth parasite, protease inhibitor, protein evolution, protein structure, stefin,
• MeSH
• cystatiny * genetika   chemie   MeSH
• disulfidy MeSH
• Fasciola hepatica * genetika   MeSH
• fylogeneze MeSH
• proteiny červů chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystatiny * MeSH
• disulfidy MeSH
• proteiny červů MeSH

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes, it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of nonoptimal peptide substrates. Finally, our homology modelling and docking studies provide potential structural explanations of the differences in specificity between DPAP1, DPAP3, and CatC, and between substrates and inhibitors in the case of DPAP3. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

• Klíčová slova
• dipeptidyl aminopeptidase, malaria, positional scanning, proteases, specificity,
• MeSH
• aminokyseliny chemie   MeSH
• dipeptidylpeptidasy a tripeptidylpeptidasy metabolismus   MeSH
• erytrocyty účinky léků   metabolismus   parazitologie   MeSH
• inhibitory proteas farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• peptidové fragmenty metabolismus   MeSH
• Plasmodium falciparum účinky léků   růst a vývoj   metabolismus   MeSH
• substrátová specifita MeSH
• tropická malárie farmakoterapie   metabolismus   parazitologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• dipeptidyl peptidase III MeSH Prohlížeč
• dipeptidylpeptidasy a tripeptidylpeptidasy MeSH
• inhibitory proteas MeSH
• peptidové fragmenty MeSH

BACKGROUND: Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite. RESULTS: A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found. CONCLUSIONS: We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.

• MeSH
• Acaridae enzymologie   imunologie   MeSH
• alergeny chemie   imunologie   izolace a purifikace   MeSH
• alergie krev   imunologie   MeSH
• alfa-amylasy chemie   imunologie   izolace a purifikace   MeSH
• feces chemie   MeSH
• hmyzí proteiny chemie   imunologie   izolace a purifikace   MeSH
• imunoglobulin E krev   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• strukturní homologie proteinů MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• zkřížené reakce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alergeny MeSH
• alfa-amylasy MeSH
• hmyzí proteiny MeSH
• imunoglobulin E MeSH

Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

• MeSH
• cysteinové endopeptidasy metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• hemoglobiny chemie   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• katalytická doména MeSH
• kathepsin B metabolismus   MeSH
• kathepsin C metabolismus   MeSH
• kathepsin D metabolismus   MeSH
• kathepsin L metabolismus   MeSH
• klíště enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• molekulární sekvence - údaje MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• asparaginylendopeptidase MeSH Prohlížeč
• cysteinové endopeptidasy MeSH
• endopeptidasy MeSH
• hemoglobiny MeSH
• inhibitory enzymů MeSH
• kathepsin B MeSH
• kathepsin C MeSH
• kathepsin D MeSH
• kathepsin L MeSH

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.

• MeSH
• acetáty farmakologie   MeSH
• cyklopentany farmakologie   MeSH
• geneticky modifikované rostliny MeSH
• inhibitory proteas metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• listy rostlin metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• oxylipiny MeSH
• posttranslační úpravy proteinů MeSH
• proteasy metabolismus   MeSH
• proteinové prekurzory metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• stabilita enzymů MeSH
• tabák enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetáty MeSH
• cyklopentany MeSH
• inhibitory proteas MeSH
• methyl jasmonate MeSH Prohlížeč
• oxylipiny MeSH
• proteasy MeSH
• proteinové prekurzory MeSH
• rostlinné proteiny MeSH