Extracellular Metalloprotease Research Articles

Effect of arazyme on the lipopolysaccharide-induced inflammatory response in human endothelial cells

Arazyme is a novel extracellular metalloprotease secreted by Aranicolaproteolyticus. Endothelial cells are involved in the pathogenesis of a number of inflammatory diseases, induce uncontrolled cell viability and express various inflammatory mediators, including cytokines, chemokines, adhesion molecules and reactive oxygen species (ROS). In the current study, human umbilical vein endothelilal cells (HUVECs) were used to investigate the anti‑inflammatory effects of arazyme following lipopolysaccharide (LPS) stimulation. Apoptosis of HUVECs due to LPS was inhibited by arazyme. In various inflammatory responses induced by LPS, arazyme inhibited the secretion of the monocyte chemoattractant protein‑1 and interleukin‑6, and the expression of vascular cell adhesion molecule‑1 and intercellular adhesion molecule‑1. Arazyme also suppressed ROS production in HUVECs. The action of arazyme was not associated with NF‑κB activity in HUVECs. These results indicate that arazyme has anti‑inflammatory properties in inflamed endothelial cells and may be useful as a therapeutic agent for inflammatory diseases associated with endothelial cells.

Abstract 353: Triciribine, a Selective Akt Inhibitor, Inhibits Myofibroblast Differentiation and Ameliorates Pulmonary Vascular Remodeling in Pulmonary Hypertension

Objectives: Idiopathic pulmonary fibrosis (IPF) is an incurable, chronic and progressive disease with severely poor prognosis and often leads to pulmonary hypertension (PH). Persistent myofibroblast (MF) differentiation in response to TGFβ, marked by de novo expression of αSMA stress fibers, is the central orchestrator of tissue fibrosis and vasculopathy. Our previous studies indicated the integral role of Akt in myofibroblast differentiation and fibrosis. Here, we investigated the efficacy of Triciribine (TCBN), a selective Akt inhibitor, currently in clinical trials for cancer therapy, as a potential therapeutic option for IPF and PH. Methods: WT and Akt1 null mice, as well as those treated with TCBN were subjected to 3 week hypoxia along with normoxia control, and the lungs were used for imaging and western analysis to study the biochemical, structural and vascular changes in the lungs. Cutting edge techniques such as microfil-cast based vascular imaging, confocal imaging and analysis of extracellular matrix protein turnover was performed for the data collection and analysis. Results: Under hypoxia, WT mice exhibited robust vascular remodeling leading to thickening of the artery wall, reduction in lumen size and loss of capillaries in the lungs. This effect was significantly less in Akt1 null mice. Treatment with TCBN significantly ameliorated vascular remodeling in mice due to hypoxia, and resulted in normalization of the vasculature with increased lumen size, reduced wall thickness and increased number of capillary branches as compared to untreated controls. TCBN also affected expression of a number of different extracellular matrix proteins and matrix metalloproteases in addition to changes in the expression of contractile proteins alpha-actin, myocardin and serum response factor in the adventitial fibroblasts. Conclusions: Our results demonstrate the crucial role of Akt as an integral mediator of MF differentiation in IPF and PH. Further, TCBN could potentially be a therapeutic option for IPF and PH.

Abstract 272: Targeted Activatable Cell-Penetrating Peptides Identify Atherosclerotic Plaques in Mice with Noninvasive Magnetic Resonance Imaging

Atherosclerotic plaque progression from stable lesions to advanced plaques that can spontaneously disrupt is promoted by numerous enzymes, including thrombin and extracellular matrix metalloproteases (MMPs). Assessing the activity of these enzymes in vivo by noninvasive MRI could be a valuable tool for the risk assessment of plaque disruption and a powerful method for monitoring the effectiveness of therapies. Novel thrombin- and MMP-targeted activatable cell-penetrating peptides (ACPPs) have a mechanism for activation and retention at sites of high enzymatic activity and show great promise for plaque detection due to incorporation of gadolinium (Gd), an MR active agent. In addition to the morphological information that is traditionally provided by MRI, both ACPPs also allow for a functional assessment of plaque. We hypothesized that these new targeted ACPPs would provide enhanced contrast at a smaller dosage and improve the sensitivity and specificity of plaque detection. The aorta of fat fed male ApoE knockout mice were imaged with a T1-weighted spin echo sequence before and after the administration of a substandard dose of non-targeted Gd-DTPA (2.5 μmol/kg, compared to the standard dose of 0.1-0.2 mol/kg). Administration of a comparable dose of thrombin- or MMP-targeted ACPP yielded a great increase in contrast around the aortic plaques (Figure 1). In conclusion, administration of the targeted ACPP achieved superior contrast and, therefore, demonstrates the potential to achieve high detection power of plaques with a much lower and safer dose of Gd.

Discovery of the first potent and selective Mycobacterium tuberculosis Zmp1 inhibitor

The Mycobacterium tuberculosis extracellular zinc metalloprotease 1 (Zmp1) has been proposed to play a key role in phagosome maturation and to enhance the survival of Mycobacterium tuberculosis in the host. Consequently, small molecule inhibitors of Zmp1 are of pivotal importance as a tool to better understand the pathogenicity of Zmp1 and as lead candidates for pharmacological intervention. Here we combined in silico structure-based inhibitor design with biochemical studies to discover and characterize the first potent competitive Zmp1 inhibitor showing a Ki of 94nM and a high selectivity for Zmp1 with respect to human Neprilysin.

Low dietary zinc promotes cell stress following blast injury (122.5)

Increased metallothionein (MT) and extracellular matrix metalloprotease (MMP) activity has been documented in skeletal muscle following ischemia/reperfusion injury (I/R). MTs bind zinc (Zn) and MMPs, possibly affecting activation of the Zn‐dependent mTOR pathway and MMP‐dependent tissue remodeling post‐injury. Because dietary Zn can reduce I/R, we evaluated the effects of 5‐weeks of low‐ and adequate Zn diets on MMP and TIMP expression in muscle following exposure to blast. Thirty‐two anesthetized Wistar rats were exposed to explosive blast (18g Semtex; 120kPa incident pressure); 32 rats served as Controls. Soleus muscle was harvested at 48h and 14d post‐blast. qRT‐PCR was used to quantify mRNA. All blast‐exposed rats exhibited transient bradycardia and bradypnea post‐injury. Loss‐of‐consciousness averaged 200 sec. At 48h post‐injury, there was a blast‐induced (P<0.05), and Zn‐responsive (P<0.05) upregulation of MT‐MMP (4.3‐fold), MMP‐2 (3.0‐fold), TIMP‐1 (5.6‐fold) and TIMP‐2 (3.4‐fold). MMP‐3 and MMP‐9 were inhibited by the blast, and were not responsive to Zn‐supplementation. No differences were observed 14d post‐blast. Our results suggest that exposure to blast activates MMPs acutely in skeletal muscle, and this response is attenuated by 14d. Low Zn diets interfere with the MMP/TIMP response in muscle, suggesting that Zn‐supplementation ameliorates blast‐induced cell stress.Grant Funding Source: Supported by US Army MRMC, US Air Force MSA

Proteolytic Processing of the Extracellular Scaffolding Protein LEV-9 Is Required for Clustering Acetylcholine Receptors

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.

Identification of a Novel Zinc Metalloprotease through a Global Analysis of Clostridium difficile Extracellular Proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.

Extracellular Novel Metalloprotease from Xenorhabdus indica and Its Potential as an Insecticidal Agent

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

Regulation system of serine protease production in Vibrio vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in cultured eels or shrimps, as well as in immunocompromised humans. An extracellular metalloprotease has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA. In the present study, we first clarified the regulatory characteristics of the VvsA production in V. vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel. V. vulnificus coordinates expression of virulence genes in response to the bacterial cell density, which is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). When cultivated at 26°C, the vvsA expression was closely related with expression of the luxS gene encoding the synthase of the AI-2 precursor LuxS. Both VvsA and AI-2 were sufficiently secreted at early stationary phase of the bacterial growth. In contrast, when cultivated at 37°C, far less amounts of the AI-2 and VvsA were produced even at the stationary phase. Disruption of the luxS gene was found to decrease significantly the vvsA expression and VvsA production. Disruption of luxO encoding the central response regulator of the QS circuit caused an increase in the vvsA expression and VvsA production at the logarithmic growth phase. These findings indicate that VvsA production is positively regulated by the V. vulnificus LuxS-dependent QS system, which operated more effectively at 26°C than at 37°C.

English

We partially purified extracellular metalloprotease of a halotolerant alkaliphilic bacterium isolated from coastal sediment of Goa, India, which was identified as Bacillus cereus strain CS1 based on biochemical characteristics and 16S rDNA sequence. Zymogram of partially purified enzyme clearly revealed two prominent protease isozymes of 210 and 215 kDa. Although, protease showed activity over a wide range of pH (pH 8 to 12) and temperature (30, 37, 45 and 50°C), optimum pH and temperature were 9 and 37°C, respectively. Protease exhibited extreme halotolerance upto 8% NaCl making it one of the industrially valuable enzyme. Among different carbon sources, glucose (1%) enhanced maximum enzyme activity (71U/ml), whereas, among the nitrogen sources, tryptone (0.2%) proved best to enhance enzyme activity (158U/ml). Ca++ ions enhanced protease activity by 7%, whereas, Cu ++ , Mg ++ , Pb ++ and Hg ++ ions inhibited activity by 91, 79, 87 and 77%, respectively. Among inhibitors tested, protease activity was significantly inhibited by Na2–EDTA as residual enzyme activity was only 21%, which clearly confirmed it to be a metalloprotease. The partially purified protease mixed separately with two detergents Rin and Wheel clearly demonstrated significant stain removing capability. Thus, protease produced by Bacillus is a potential candidate as an additive in detergent industry. Keywords: Halo-tolerant, isozymes, zymogram, metalloprotease. African Journal of Biotechnology Vol. 12(31), pp. 4905-4914

Overexpression of Matrix Metalloproteinase 11 in Thai Prostatic Adenocarcinoma is Associated with Poor Survival

The incidence of prostate cancer, one of the most common cancers in elderly men, is increasing annually in Thailand. Matrix metalloproteinase 11 (MMP-11) is a member of the extracellular matrix metalloproteases which has been associated with human tumor progression and clinical outcome. To quantify MMP-11 expression in prostatic adenocarcinoma tissues and to determine whether its overexpression correlates with survival outcome, and to assess its potential as a new prognostic marker. Expression of MMP-11 was analyzed using immunohistochemistry in 103 Thai patients with prostatic adenocarcinoma. Overall survival was analyzed using Kaplan-Meier methods and Cox regression models. Immunoreactivity of MMP-11 was seen in the stroma of prostatic adenocarcinoma tissue samples, high expression being significantly correlated with poor differentiation in Gleason grading, pathologic tumor stage 4 (pT4), and positive-bone metastasis (p<0.05), but not age and prostatic-specific antigen (PSA) level. Patients with high levels of MMP-11 expression demonstrated significantly shorter survival (p<0.001) when compared to those with low levels. Multivariate analysis showed that MMP-11 expression and pT stage were related with survival in prostatic adenocarcinoma [hazard ratio (HR)=0.448, 95% confidence interval (95%CI)=0.212-0.946, HR=0.333, 95%CI=0.15-0.74, respectively]. Expression of MMP-11 is significantly associated with survival in prostatic adenocarcinoma. High levels may potentially be used for prediction of a poor prognosis.

Abstract TP79: Imbalance Between Activation Of Matrix Metalloprotease And Its Inhibition May Facilitate The Growth Of Cerebral Aneurysm In Female Rats

Introduction: Cerebral aneurysms are a major cause of subarachnoid hemorrhage . Despite the catastrophic consequences of their rupture, not all mechanisms underlying the formation, progression, and rupture of cerebral aneurysms are fully understood. Using our model of reproducible cerebral aneurysms in oophorectomized rats we have documented the pathogenesis of cerebral aneurysms. Methionine-induced hyperhomocysteinemia which participates in coronary disease or stroke, is also associated with cerebral aneurysm formation. Whether it facilitates the growth and rupture of cerebral aneurysms remains obscure. We explored the role of methionine in the growth and rupture of cerebral aneurysms. Methods: We subject 13 week-old female Sprague-Dawley rats fed an 8% high-salt diet to bilateral oophorectomy and ligation of the right common carotid artery. Four weeks later they underwent ligation of the bilateral posterior renal artery divided into 2 groups; one group did and the other did not receive 1g/kg/day methionine in the drinking water. The aneurysmal rupture rate and morphologic- and genomic changes in the 2 groups were compared at 12 weeks. Results: The incidence of cerebral aneurysm formation was the same in both groups. While the rate of rupture was not higher in the methionine group, their aneurysms were larger and oxidative stress-related NOX4, Rac1, and apoptosis-related Rac1 were up-regulated. Interestingly, the expression of MMP-9 was increased as was the ratio of MMP-9 to TIMP2 but not of MMP-9 to TIMP1, suggesting vascular degradation due to an imbalance between the activation and inhibition of extracellular matrix metalloprotease. Conclusion: Our findings suggest that excessive vascular degradation due to oxidative stress and apoptosis induced by methionine contribute to the progression of cerebral aneurysms in oophorectomized rats.

Structure of gentlyase, the neutral metalloprotease of Paenibacillus polymyxa.

Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1-2 and a deletion near site Ca3.

Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel β-strands with a unique fold that has a compact β-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe3+). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.

1H,15N and13C Backbone Assignments and Secondary Structures of C-ter100 Domain of Vibrio Extracellular Metalloprotease Derived from Vibrio vulnificus

Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883, KoreaReceived June 26, 2012, Accepted July 6, 2012Vibrio extracellular metalloprotease (vEP), secreted from Vibrio vulnificus , shows various proteolytic functionsuch as prothrombin activation and fibrinolytic activities. Premature form of vEP has an N-terminal (nPP) anda C-terminal (C-ter100) re gion. The nPP and C-ter100 regions are autocl eaved for the matu red metalloproteaseactivity. It has been proposed that two regions play a key role in regulating enzymatic activity of vEP.Especially, C-ter100 has a regulatory function on proteolytic activity of vEP. C-ter100 domain has been clonedinto the E. coli expression vectors, pET32a and pG EX 4T-1 with TEV pr otease cleav age site and pu rified usinggel-filtration chromatography followed by affinity chromatography. To understand how C-ter100 modulatesproteolytic activity of vEP, structural studies were performed by heteronuclar multi-dimensional NMRspectroscopy. Backbone

Sizzled-Tolloid Interactions Maintain Foregut Progenitors by Regulating Fibronectin-Dependent BMP Signaling

The liver, pancreas, and lungs are induced from endoderm progenitors by a series of dynamic growth factor signals from the mesoderm, but how the temporal-spatial activity of these signals is controlled is poorly understood. We have identified an extracellular regulatory loop required for robust bone morphogenetic protein (BMP) signaling in the Xenopus foregut. We show that BMP signaling is required to maintain foregut progenitors and induce expression of the secreted frizzled related protein Sizzled (Szl) and the extracellular metalloprotease Tolloid-like 1 (Tll1). Szl negatively regulates Tll activity to control deposition of a fibronectin (FN) matrix between the mesoderm and endoderm, which is required to maintain BMP signaling. Foregut-specific Szl depletion results in a loss of the FN matrix and failure to maintain robust pSmad1 levels, causing a loss of foregut gene expression and organ agenesis. These results have implications for BMP signaling in diverse contexts and the differentiation of foregut tissue from stem cells.

A Unique Regulator Contributes to Quorum Sensing and Virulence in Burkholderia cenocepacia

Burkholderia cenocepacia causes chronic and life-threatening respiratory infections in immunocompromized people. The B. cenocepacia N-acyl-homoserine lactone (AHL)-dependent quorum sensing system relies on the production of AHLs by the synthases CepI and CciI while CepR, CciR and CepR2 control expression of many genes important for pathogenesis. Downstream from, and co-transcribed with cepI, lies BCAM1871 encoding a hypothetical protein that was uncharacterized prior to this study. Orthologs of B. cenocepacia BCAM1871 are uniquely found in Burkholderia spp and are conserved in their genomic locations in pathogenic Burkholderia. We observed significant effects on AHL activity upon mutation or overexpression of BCAM1871, although these effects were more subtle than those observed for CepI indicating BCAM1871 acts as an enhancer of AHL activity. Transcription of cepI, cepR and cciIR was significantly reduced in the BCAM1871 mutant. Swimming and swarming motilities as well as transcription of fliC, encoding flagellin, were significantly reduced in the BCAM1871 mutant. Protease activity and transcription of zmpA and zmpB, encoding extracellular zinc metalloproteases, were undetectable in the BCAM1871 mutant indicating a more significant effect of mutating BCAM1871 than cepI. Exogenous addition of OHL restored cepI, cepR and fliC transcription but had no effect on motility, protease activity or zmpA or zmpB transcription suggesting AHL-independent effects. The BCAM1871 mutant exhibited significantly reduced virulence in rat chronic respiratory and nematode infection models. Gene expression and phenotypic assays as well as vertebrate and invertebrate infection models showed that BCAM1871 significantly contributes to pathogenesis in B. cenocepacia.

Serum levels of ADAM12‐S: possible association with the initiation and progression of dermal fibrosis and interstitial lung disease in patients with systemic sclerosis

A disintegrin and metalloprotease (ADAM) 12 is one of the metalloproteinase-type ADAMs and possesses extracellular metalloprotease and cell-binding functions. ADAM12 is expressed in two alternative forms, such as a membrane-anchored form (ADAM12-L) and a short secreted form (ADAM12-S). To investigate the clinical significance of serum ADAM12-S levels in systemic sclerosis (SSc). Serum ADAM12-S levels were determined by a specific enzyme-linked immunosorbent assay in 61 SSc patients and 18 healthy controls. Serum ADAM12-S levels were significantly increased in diffuse cutaneous SSc (dcSSc) patients than in healthy controls (0.417 ± 0.389 vs. 0.226 ± 0.065 ng/mL; P < 0.05), while being comparable between limited cutaneous SSc (0.282 ± 0.258 ng/mL) and healthy controls. Serum ADAM12-S levels significantly elevated in dcSSc patients with disease duration of ≤ 6 years (0.537 ± 0.449 ng/mL, P < 0.05), but not in dcSSc with disease duration of >6 years (0.225 ± 0.049 ng/mL), compared to healthy controls. Furthermore, in dcSSc patients with disease duration of ≤ 6 years, serum ADAM12-S levels correlated positively with modified Rodnan total skin thickness score, ground glass score, and serum C-reactive protein values, while showed inverse correlation with fibrosis score. Elevated serum ADAM12-S levels are associated with elevated serum inflammatory marker, severity of skin fibrosis, and activity of interstitial lung disease in dcSSc, suggesting the possible contribution of ADAM12-S to the pathological events in this disorder.

Abstract 806: Extracellular matrix metalloprotease inducer (EMMPRIN) and CD44 protein complexes are exclusively formed in basal- and normal-like breast cancer cell lines

Abstract Extracellular Matrix Metalloprotease Inducer (EMMPRIN) is a multifunctional glycoprotein that is involved in tumor cell invasion and metastasis, and is a predictor for tamoxifen resistance in breast cancer. EMMPRIN is implicated in development of therapy resistance by forming a functional transporter complex through interaction with CD44 and monocarboxylate transporter 4. Presence of EMMPRIN transporter protein complexes may therefore be a better predictor for therapy resistance and a direct target for drugs. In this study, we aimed to identify EMMPRIN-CD44 protein interactions in a large panel of breast cancer cell lines. EMMPRIN-CD44 protein interaction was investigated in a panel of 41 breast cancer cell lines, which represent the luminal (n=12), ErbB2 (n=13), basal (n=7), and normal-like (n=9) intrinsic subtypes, using in situ Proximity Ligation Assay (isPLA). In addition, protein expression of both EMMPRIN and CD44 in the cell lines was evaluated by immunohistochemistry (IHC). IHC and isPLA read-out was performed by bright field microscopy and 0-1-2-3 scoring was applied to none-weak-moderate-strong staining (IHC) or interaction (isPLA), respectively. Moderate and strong EMMPRIN expression was observed in 50% of luminal, 36% of ErbB2, 86% of basal, and 100% of normal-like cell lines, and moderate to high CD44 expression was detected in 64%, 83%, 86%, and 100% in luminal, ErbB2, basal, and normal-like cell lines, respectively. Interestingly, moderate to strong EMMPRIN-CD44 interaction was exclusively detected in basal (64%) and normal-like (67%) cell lines, whereas luminal and ErbB2 cells lines had no detectible, or only very weak, EMMPRIN-CD44 interaction (≥square = 23.5, P&amp;lt;0.0001). Our results indicate that EMMPRIN-CD44 protein complexes are formed exclusively in basal and normal-like breast cancer cell lines. These breast cancer subtypes are associated with an aggressive phenotype and poor patient survival. We speculate that active EMMPRIN-CD44 transporters are involved in drug resistance, resulting in poor clinical outcome, and will investigate this in more detail in clinical breast cancer specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 806. doi:1538-7445.AM2012-806

The orphan histidine protein kinase SgmT is a c‐di‐GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.