Reactive Site Loop Research Articles

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 μg/ml) showed a 2–4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition.

SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this study was to generate antibodies directed against different SerpinB3 epitopes to better investigate their biological role. Five exposed epitopes were identified using the software DNASTAR Lasergene and the corresponding synthetic peptides were used for NZW rabbit immunization. Anti-P#2 and anti-P#4 antibodies were able to recognize both SerpinB3 and SerpinB4 by ELISA. Anti-P#5 antibody, produced against the reactive site loop of SerpinB3, showed the greatest specific reactivity for human SerpinB3. This antibody was able to recognize SerpinB3 at nuclear level, while anti-P#3 antibody recognized SerpinB3 only at cytoplasmic level, both by immunofluorescence and by immunohistochemistry. The biological activity of each antibody preparation was assessed in HepG2 cells overexpressing SerpinB3 and anti-P#5 antibody reduced proliferation by 12% cell and cell invasion by 75%, while trivial results were obtained with the other antibody preparations. These findings indicate that the reactive site loop of SerpinB3 is essential for the invasiveness features induced by this serpin and it could become a novel druggable target.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Scaffold stability and P14' residue steric hindrance in the differential inhibition of FXIIa by Aedes aegypti trypsin inhibitor versus Infestin-4.

Kazal-type protease inhibitors strictly regulate Factor XIIa (FXIIa), a blood-clotting serine protease. However, when negatively charged surface of prosthetic device come into contact with FXII, it undergoes conformational change and auto-activation, leading to thrombus formation. Some research suggests that Kazal-type protease inhibitor specificity against FXIIa is governed solely by the reactive-site loop sequence, as this sequence makes most-if not all-of the direct contacts with FXIIa. Here, we sought to compare the inhibitory properties of two Kazal-type inhibitors, Infestin-4 (Inf4), a potent inhibitor of FXIIa, and Aedes aegypti trypsin inhibitor (AaTI), which does not inhibit FXIIa, to better understand Kazal-type protease specificity and determine the structural components responsible for inhibition. There are only three residue differences in the reactive-site loop between AaTI and Inf4. Through site-directed mutagenesis, we show that the reactive-site loop is only partially responsible for the inhibitory specificity of these proteases. The protein scaffold of AaTI is unstable due to an elongated C5C6 region. Through chimeric study, we show that swapping the protease-binding loop and the C5C6 region from Inf4 with that of AaTI can partially enhance the inhibitory activity of the AaTI_Inf4 chimera. Furthermore, the additional substitution of Asn at the P14′ position of AaTI with Gly (Gly27 in Inf4) absolves the steric clashing between AaTI and the surface 140-loop of FXIIa, and increases the inhibition of the chimeric AaTI to match that of wild-type Inf4. Our findings suggest that ancillary regions in addition to the reactive-site loop sequence are important factors driving Kazal-type inhibitor specificity.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Crystal structure of Aedes aegypti trypsin inhibitor in complex with μ-plasmin reveals role for scaffold stability in Kazal-type serine protease inhibitor.

Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.

Isolation and functional diversity of Bowman–Birk type serine proteinase inhibitors from Hyacinthus orientalis

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Antithrombin Phe229Leu: a new homozygous variant leading to spontaneous antithrombin polymerization in vivo associated with severe childhood thrombosis

AbstractThere is increasing evidence that serpin conformational alteration caused by single point mutations can be responsible for protein deficiency associated with human diseases. A typical example is the α1-antitrypsin deficiency caused by the Z variant carrying a Glu342Lys substitution. Only a few cases of “conformational disease” involving other serpins have been described so far. We investigated a severe antithrombin deficiency in a 13-month-old child with fever and cerebral venous thrombosis. The infant was found to be homozygous for a new antithrombin gene mutation (7396T>C, predicting a Phe229Leu antithrombin variant), and heterozygous for the factor V Leiden mutation. Mild atypical antithrombin deficiency was found in both parents, who were first cousins, asymptomatic, and heterozygous for the same antithrombin gene mutation. The Phe229Leu variant, which does not readily fit into the current classification of antithrombin deficiency, was shown to be a thermolabile antithrombin that spontaneously polymerized in the proband's circulation. This points to a key role for the conserved Phe at position 229, which is near the reactive site loop in a region critical for serpin function and stability. Molecular modeling suggested how the mutation might destabilize this region of the protein and thereby favor reactive site loop insertion and polymerization. This study provides the first direct evidence of antithrombin polymerization in vivo causing antithrombin deficiency and severe thrombotic disease.

Bioengineering of an elastase inhibitor from Caesalpinia echinata (Brazil wood) seeds

Protease inhibitors have been widely used in several therapeutic applications such as in the treatment of bleeding disorders, hypertension, cancer and pulmonary diseases. In a previous work, we demonstrated that a Kunitz-type serine protease inhibitor isolated from the seeds of Caesalpinia echinata (CeEI) exhibits pharmacological potential in lung inflammatory diseases in which neutrophil elastase plays a crucial role. However, an important challenge in the use of natural products is to ensure a commercially viable production. In this work, we report the cloning, expression and purification of two recombinant CeEI isoinhibitors with 700 base pairs encoding two proteins with 181 amino acid residues (rCeEI-4 and rCeEI-5). After the expression, each yielding 22 mg/L of active protein, both isoinhibitors presented a molecular mass of about 23.0 kDa, evaluated by SDS-PAGE. The inhibition constants for human neutrophil elastase (HNE) were 0.67 nM (rCeEI-4) and 0.57 nM (rCeEI-5), i.e., similar to the native inhibitor (1.90 nM). Furthermore, rCeEI-4 was used as a template to design smaller functional peptides flanking the inhibitor reactive site: rCeEI-36, delimited between the amino acid residues N36 and S88 containing a disulfide bond in the reactive-site loop, and rCeEI-46, delimited between S46 and L75 without the disulfide bond. The yields were 18 mg/L (rCeEI-36) and 12 mg/L (rCeEI-46). Both peptides inhibit HNE in the nanomolar range (Ki 0.30 ± 0.01 and 8.80 ± 0.23, respectively). Considering their size and the inhibitory efficiency, these peptides may be considered in strategies for the development of drugs targeting pulmonary disorders where elastase is involved.

An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica

Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants < 10 nM). Their unusual inhibitory properties are due to (a) Leu15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1′ subsites of the cathepsin protease, and (b) Arg19 which forms cation-π interactions with Trp291 of the S1′ subsite and electrostatic interactions with Asp125 of the S2′ subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu15 in the reactive loop with Arg15. The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.

Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.

Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

NMR structure of CmPI-II, a non-classical Kazal protease inhibitor: Understanding its conformational dynamics and subtilisin A inhibition

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2′ positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2′) contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

Homologous Lympho-Epithelial Kazal-type Inhibitor Domains Delay Blood Coagulation by Inhibiting Factor X and XI with Differential Specificity

Despite being initially identified in the blood filtrate, LEKTI is a 15-domain Kazal-type inhibitor mostly known in the regulation of skin desquamation. In the current study, screening of serine proteases in blood coagulation cascade showed that LEKTI domain 4 has inhibitory activity toward only FXIa, whereas LEKTI domain 6 inhibits both FXIa and FXaB (bovine FXa). Nuclear magnetic resonance structural and dynamic experiments plus molecular dynamics simulation revealed that LEKTI domain 4 has enhanced backbone flexibility at the reactive-site loop. A model of the LEKTI-protease complex revealed that FXaB has a narrower S4 pocket compared with FXIa and hence prefers only small side-chain residues at the P4 position, such as Ala in LEKTI domain 6. Mutational studies combined with a molecular complex model suggest that both a more flexible reactive-site loop and a bulky residue at the P4 position make LEKTI domain 4 a weaker but highly selective inhibitor of FXIa.

Docking, thermodynamics and molecular dynamics (MD) studies of a non-canonical protease inhibitor, MP-4, from Mucuna pruriens

Sequence and structural homology suggests that MP-4 protein from Mucuna pruriens belongs to Kunitz-type protease inhibitor family. However, biochemical assays showed that this protein is a poor inhibitor of trypsin. To understand the basis of observed poor inhibition, thermodynamics and molecular dynamics (MD) simulation studies on binding of MP-4 to trypsin were carried out. Molecular dynamics simulations revealed that temperature influences the spectrum of conformations adopted by the loop regions in the MP-4 structure. At an optimal temperature, MP-4 achieves maximal binding while above and below the optimum temperature, its functional activity is hampered due to unfavourable flexibility and relative rigidity, respectively. The low activity at normal temperature is due to the widening of the conformational spectrum of the Reactive Site Loop (RSL) that reduces the probability of formation of stabilizing contacts with trypsin. The unique sequence of the RSL enhances flexibility at ambient temperature and thus reduces its ability to inhibit trypsin. This study shows that temperature influences the function of a protein through modulation in the structure of functional domain of the protein. Modulation of function through appearance of new sequences that are more sensitive to temperature may be a general strategy for evolution of new proteins.

115 - Plant-Based Production, Purification, and Characterization of Oxidation-Resistant Alpha-1 Antitrypsin

Alpha-1 antitrypsin (AAT) is an acute phase protease inhibitor. Currently, the only available treatment for patients with COPD caused by AAT deficiency is replacement therapy, requiring weekly infusions of human plasma-derived AAT. While AAT has shown potential when inhaled to treat both emphysema and cystic fibrosis, the oxidation susceptibility of methionine residues at positions 351 and 358 in the reactive site loop have led to difficulties in maintaining AAT activity and stability when aerosolized. Using a novel cucumber mosaic virus-based inducible transient expression system, we have produced a biobetter plant recombinant AAT (prAAT) in Nicotiana benthamiana . This variant replaces the oxidation-susceptible methionine residue at position 358 with a valine residue to increase resistance to oxidation. N. benthamiana plants were vacuum infiltrated as detached leaves with two strains of Agrobacterium tumefaciens : one carrying the biobetter AAT gene and one carrying a gene for the tomato bushy stunt virus p19 viral RNA silencing suppressor driven by a constitutive cauliflower mosaic virus 35S promoter. Leaves were incubated in humidity chambers at 20°C in the dark for 6 days before flash freezing in liquid nitrogen, grinding, and protein extraction in a 20 mM Tris, 150 mM NaCl, and 0.01% (v/v) Tween-80 AAT stability buffer. Purification was achieved using Alpha-1 Antitrypsin Select Affinity Chromatography (GE Healthcare) with a 58.1±8.1% yield of active AAT. Activity of purified AAT was confirmed via a band shift in a Western blot after incubation with excess porcine pancreatic elastase, however, degradation of prAAT was also observed in all purified extracts. Purified prAAT was tested against an analytical standard (Calbiochem) and Prolastin-C (Griffols), a common therapeutic formulation of human AAT, to determine its activity and oxidation resistance. 41±11% of purified prAAT and 71.9±5.5% of Prolastin-C showed activity against porcine pancreatic elastase as determined by a residual elastase activity inhibitory assay. Under oxidation with 48.9 mM H 2 O 2 for 60 minutes at room temperature, prAAT was found to retain 102.8±20.4% of its original anti-elastase activity, while Prolastin-C retained 13.8±5.0% anti-elastase activity and analytical standard AAT retained 11.6±7.5% anti-elastase activity. Future work will include determinations of oxidation resistance to OCl – /HOCl and development of more optimal isolation and purification processes.

Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins

Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.

Loss-of-Function Mutations in SERPINB8 Linked to Exfoliative Ichthyosis with Impaired Mechanical Stability of Intercellular Adhesions

SERPINS comprise a large and functionally diverse family of serine protease inhibitors. Here, we report three unrelated families with loss-of-function mutations in SERPINB8 in association with an autosomal-recessive form of exfoliative ichthyosis. Whole-exome sequencing of affected individuals from a consanguineous Tunisian family and a large Israeli family revealed a homozygous frameshift mutation, c.947delA (p.Lys316Serfs(∗)90), and a nonsense mutation, c.850C>T (p.Arg284(∗)), respectively. These two mutations are located in the last exon of SERPINB8 and, hence, would not be expected to lead to nonsense-mediated decay of the mRNA; nonetheless, both mutations are predicted to lead to loss of the reactive site loop of SERPINB8, which is crucial for forming the SERPINB8-protease complex. Using Sanger sequencing, a homozygous missense mutation, c.2T>C (p.Met1?), predicted to result in an N-terminal truncated protein, was identified in an additional family from UAE. Histological analysis of a skin biopsy from an individual homozygous for the variant p.Arg284(∗) showed disadhesion of keratinocytes in the lower epidermal layers plus decreased SERPINB8 levels compared to control. Invitro studies utilizing siRNA-mediated knockdown of SERPINB8 in keratinocytes demonstrated that in the absence of the protein, there is a cell-cell adhesion defect, particularly when cells are subjected to mechanical stress. In addition, immunoblotting and immunostaining revealed an upregulation of desmosomal proteins. In conclusion, we report mutations in SERPINB8 that are associated with exfoliative ichthyosis and provide evidence that SERPINB8 contributes to the mechanical stability of intercellular adhesions in the epidermis.

Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense.