Matrix metalloproteinase-8 deficiency promotes granulocytic allergen-induced airway inflammation

Matrix metalloproteinases (MMPs) are involved in inflammatory reaction, including asthma-related airway inflammation. MMP-8, mainly produced by neutrophils, has recently been reported to be increased in the bronchoalveolar lavage fluid (BALF) from asthmatic patients. To evaluate the role of MMP-8 in asthma, we measured MMP-8 expression in lung tissue in an OVA-sensitized mouse model of asthma and addressed the effect of MMP-8 deletion on allergen-induced bronchial inflammation. MMP-8 production was increased in lungs from C57BL/6 mice exposed to allergens. After allergen exposure, MMP-8(-/-) mice developed an airway inflammation characterized by an increased neutrophilic inflammation in BALF and an increased neutrophilic and eosinophilic infiltration in the airway walls. MMP-8 deficiency was associated with increased levels of IL-4 and anti-OVA IgE and IgG1 in BALF and serum, respectively. Although allergen exposure induced an enhancement of LPS-induced CXC chemokine, KC, and MIP-2 levels in BALF and lung parenchyma, no difference was observed between the two genotypes. Inflammatory cell apoptosis was reduced in the lungs from MMP-8(-/-) mice. For the first time, our study evidences an important role of MMP-8 in the control of neutrophilic and eosinophilic infiltration during allergen-induced lung inflammation, and demonstrates that the anti-inflammatory effect of MMP-8 is partly due to a regulation of inflammatory cell apoptosis.     

N-terminal cysteinyl proteins can be prepared using thrombin cleavage

Expressed protein ligation--which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment--has emerged as a powerful technique. The technique requires the formation of a C-terminal alpha-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications.     

Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide

Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.     

Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine–isoleucine or glycine–leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K_m similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.     

ARIA can be released from extracellular matrix through cleavage of a heparin-binding domain

ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. Here we demonstrate that the ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2-terminal portion, but preserves the active, EGF-like domain. We also show that ARIA can be released from freshly dissociated cells from embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. We present a model of how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis.     

Retinoic acid can be produced from excentric cleavage of beta-carotene in human intestinal mucosa.

The hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.     

Mining SARS-CoV protease cleavage data using non-orthogonal decision trees: a novel method for decisive template selection

Motivation: Although the outbreak of the severe acute respiratorysyndrome (SARS) is currently over, it is expected that it willreturn to attack human beings. A critical challenge to scientistsfrom various disciplines worldwide is to study the specificityof cleavage activity of SARS-related coronavirus (SARS-CoV)and use the knowledge obtained from the study for effectiveinhibitor design to fight the disease. The most commonly usedinductive programming methods for knowledge discovery from dataassume that the elements of input patterns are orthogonal toeach other. Suppose a sub-sequence is denoted as P₂P₁P_1'P_2',the conventional inductive programming method may result ina rule like ‘if P₁ = Q, then the sub-sequence is cleaved,otherwise non-cleaved’. If the site P₁ is not orthogonalto the others (for instance, P₂, P_1' and P_2'), the predictionpower of these kind of rules may be limited. Therefore thisstudy is aimed at developing a novel method for constructingnon-orthogonal decision trees for mining protease data. Result: Eighteen sequences of coronavirus polyprotein were downloadedfrom NCBI (http://www.ncbi.nlm.nih.gov). Among these sequences,252 cleavage sites were experimentally determined. These sequenceswere scanned using a sliding window with size k to generateabout 50 000 k-mer sub-sequences (for short, k-mers). The valueof k varies from 4 to 12 with a gap of two. The bio-basis functionproposed by Thomson et al. is used to transform the k-mers toa high-dimensional numerical space on which an inductive programmingmethod is applied for the purpose of deriving a decision treefor decision-making. The process of this transform is referredto as a bio-mapping. The constructed decision trees select about10 out of 50 000 k-mers. This small set of selected k-mers isregarded as a set of decisive templates. By doing so, non-orthogonaldecision trees are constructed using the selected templatesand the prediction accuracy is significantly improved. Availability: The program for bio-mapping can be obtained byrequest to the author. Contact: z.r.yang{at}exeter.ac.uk     

Matrix metalloproteinase-1 contribution to sarcoma cell invasion

Matrix metalloproteinase-1 (MMP-1) activity has been linked to numerous disease processes from arthritis to ulcer. Its proteolytic activity has been implicated inconsistently in different steps of tumourigenesis and metastasis. The discrepancies may be attributable to our limited understanding of MMP-1 production, cellular trafficking, secretion and local activation. Specifically, regulation of MMP-1 directional delivery versus its general extracellular matrix secretion is largely unknown. Inhibition of prenylation by farnesyl transferase inhibitor (FTI-276) decreased extracellular MMP-1 and subsequently reduced invasiveness by 30%. Parallel, stable cell line RNAi knockdown of MMP-1 confirmed its role in cellular invasiveness. The prenylation agonist farnesyl pyrophosphate (FPP) partially normalized FTI-276 inhibited extracellular MMP-1 levels and invasion capacity while transiently delayed its cellular podia distribution. MMP-1 directional delivery to these structures were confirmed by combination of a MMP-1-specific fluorogenic substrate, a MMP1-Ds-Red fusion protein construct expression and DQ-collagen degradation, which demonstrated coupling of directional delivery and activation. MetaMorph analysis of cellular lamellipodia structures indicated that FTI-276 inhibited formation and delivery to these structures. Farnesyl pyrophosphate partially restored lamellipodia area but not MMP-1 delivery under the time frame investigated. These results indicate that MMP-1 directional delivery to podia structures is involved in the invasive activity of sarcoma cells, and this process is prenylation sensitive.     

Permanence can be Defended

In donation after the circulatory‐respiratory determination of death (DCDD), the dead donor rule requires that the donor be dead before organ procurement can proceed. Under the relevant limb of the Uniform Determination of Death Act 1981 (USA), a person is dead when the cessation of circulatory‐respiratory function is ‘irreversible’. Critics of current practice in DCDD have argued that the donor is not dead at the time organs are procured, and so the procurement of organs from these donors violates the dead donor rule. We offer a new argument here in defence of current DCDD practice, and, in particular, of the interpretation of the requirement of ‘irreversibility’ as permanence.     

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.     

Matrix metalloproteinase-8 mediates the unfavorable systemic impact of local irradiation on pharmacokinetics of anti-cancer drug 5-Fluorouracil

Concurrent chemoradiation with 5-fluorouracil (5-FU) is widely accepted for cancer treatment. However, the interactions between radiation and 5-FU remain unclear. Here, we evaluated the influence of local irradiation on the pharmacokinetics of 5-FU in rats. The single-fraction radiation was delivered to the whole pelvic fields of Sprague-Dawley rats after computerized tomography-based planning. 5-FU at 100 mg/kg was prescribed 24 hours after radiation. A high-performance liquid chromatography system was used to measure 5-FU in the blood. Matrix metalloproteinase-8 (MMP-8) inhibitor I was administered to examine whether or not RT modulation of 5-FU pharmacokinetic parameters could be blocked. Compared with sham-irradiated controls, whole pelvic irradiation reduced the area under the concentration versus time curve (AUC) of 5-FU in plasma and, in contrast, increased in bile with a radiation dose-dependent manner. Based on protein array analysis, the amount of plasma MMP-8 was increased by whole pelvic irradiation (2.8-fold by 0.5 Gy and 5.3-fold by 2 Gy) in comparison with controls. Pretreatment with MMP-8 inhibitor reversed the effect of irradiation on AUC of 5-FU in plasma. Our findings first indicate that local irradiation modulate the systemic pharmacokinetics of 5-FU through stimulating the release of MMP-8. The pharmacokinetics of 5-FU during concurrent chemoradiaiton therapy should be rechecked and the optimal 5-FU dose should be reevaluated, and adjusted if necessary, during CCRT.     

Matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the K/BxN serum-transfer arthritis model

Introduction  

Rheumatoid arthritis is an autoimmune disease in which joint inflammation leads to progressive cartilage and bone erosion. Matrix metalloproteinases (MMPs) implicated in homeostasis of the extracellular matrix play a central role in cartilage degradation. However, the role of specific MMPs in arthritis pathogenesis is largely unknown. The aim of the present study was to investigate the role of Mmp-8 (collagenase-2) in an arthritis model.     

Caspase-8 can be activated by interchain proteolysis without receptor-triggered dimerization during drug-induced apoptosis

Proteases of the caspase family are thought to be activated by proteolytic processing of their inactive zymogens. However, although proteolytic cleavage is sufficient for executioner caspases, a different mechanism has been recently proposed for initiator caspases, such as caspase-8, which are believed to be activated by proximity-induced dimerization. According to this model, dimerization rather than proteolytic processing is considered as the critical event for caspase-8 activation. Such a mechanism would suggest that in the absence of a dimerization platform such as the death-inducing signaling complex, caspase-8 proteolytic cleavage would result in an inactive enzyme. As several studies have described caspase-8 cleavage during mitochondrial apoptosis, we now investigated whether caspase-8 becomes indeed catalytically active in this pathway. Using an in vivo affinity labeling approach, we demonstrate that caspase-8 is activated in etoposide-treated cells in vivo in the absence of the receptor-induced death-inducing signaling complex formation. Furthermore, we show that both caspase-3 and -6 are required for the efficient activation of caspase-8. Our data therefore indicate that interchain cleavage of caspase-8 in the mitochondrial pathway is sufficient to produce an active enzyme even in the absence of receptor-driven procaspase-8 dimerization.     

Immunoglobulin cleavage by the streptococcal cysteine protease IdeS can be detected using protein G capture and mass spectrometry

The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.