Pericellular Proteolysis Research Articles

Mmp14-dependent remodeling of the pericellular-dermal collagen interface governs fibroblast survival.

Dermal fibroblasts deposit type I collagen, the dominant extracellular matrix molecule found in skin, during early postnatal development. Coincident with this biosynthetic program, fibroblasts proteolytically remodel pericellular collagen fibrils by mobilizing the membrane-anchored matrix metalloproteinase, Mmp14. Unexpectedly, dermal fibroblasts in Mmp14-/- mice commit to a large-scale apoptotic program that leaves skin tissues replete with dying cells. A requirement for Mmp14 in dermal fibroblast survival is recapitulated in vitro when cells are embedded within, but not cultured atop, three-dimensional hydrogels of crosslinked type I collagen. In the absence of Mmp14-dependent pericellular proteolysis, dermal fibroblasts fail to trigger β1 integrin activation and instead actuate a TGF-β1/phospho-JNK stress response that leads to apoptotic cell death in vitro as well as in vivo. Taken together, these studies identify Mmp14 as a requisite cell survival factor that maintains dermal fibroblast viability in postnatal dermal tissues.

QSAR modelling to predict structural features of certain sulfonamide as Urokinase-type Plasminogen Activator inhibitors

The serine hydrolase family includes serine proteases. It is essential for hydrolyzing protein peptide bonds and breaking them. The urokinase-type plasminogen activator (uPA) selectively binds to the uPAR on numerous cell types, including cancer cells. Pericellular proteolysis of cell-bound proteins requires this interaction. High uPA and uPAR levels regularly worsen cancer prognoses. Thus, small chemical active-site inhibitors that block uPA may diminish cancer cell invasion and metastasis. In compliance with Organization for Economic Corporation and Development guidelines, this research performed a complete Quantitative structure activity relationship analysis of sulfonamide compounds as Urokinase-type Plasminogen Activator inhibitors. Py-Descriptors were used for this investigation. PyDescriptor uses PyMOL standards and idioms to calculate 11,145 simple molecular descriptors. This plugin calculates molecular descriptors irrespective of molecular representation properties like atom numbering or labelling, spatial reference frame, translational and rotational invariance, etc. The investigation sought to find essential and hidden structural characteristics that regulate sulfonamide-type drugs' Urokinase-type Plasminogen Activator Inhibitory action. Twenty-eight sulfonamide chemicals are used in the Quantitative structure activity relationship study to generate statistically robust and highly predictive univariate and multivariate models. All models were thoroughly evaluated and meet several statistical parameter thresholds (e.g., R2 =0.9259–0.9280, Q2Loo= 0.8579–0.8558, Q2LMO= 0.8013–0.7865). The analysis reveal that occurance of ring carbon atoms exactly at 3 A0 from carbon atom, number of negatively charged atoms from sulphur atoms within 5 bonds, presence of hydrogen atom exactly at 3 bonds from donar atoms, presence of carbon atom exactly at 4 A0 from donar atom, presence of acceptor atom exactly at 5 A0 from sulphur atom and sum of partial charges of lipo atoms within 6 bonds from sulphur atom are important pharmacophoric features for Urokinase-type Plasminogen Activator Inhibition binding affinity. Thus, the developed Quantitative structure activity relationship study has an equilibrium of quantitative and qualitative tactics. The results could be useful for future optimizations of sulfonamide analogues.

A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue

The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with Ki values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (Ki = 0.20 µM) and murine orthologue (Ki = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.

330 (PB110) - Evaluation of membrane type 1 metalloproteinase (MT1-MMP/MMP14) expression as a prognostic biomarker in patients with solid tumours screened for a Phase I/II trial

Background: MT1-MMP plays an important role in pericellular proteolysis and is implicated in cancer cell migration, growth, invasion and angiogenesis. Limited evidence suggests that MT1-MMP overexpression is associated with a poor prognosis in numerous solid tumours. Using data from immunohistochemical pre-screening for a Phase 1/2 clinical trial targeting MT1-MMP (clinicaltrials.gov identifier: NCT03486730), we determined if MT1-MMP over-expression was a prognostic biomarker in selected solid tumours.

Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment-Peculiar Effect of Epigallocatechin-3-Gallate.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

TMPRSS13 zymogen activation, surface localization, and shedding is regulated by proteolytic cleavage within the non-catalytic stem region.

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Here we characterize a novel post-translational mechanism important for TMPRSS13 function: proteolytic cleavage within the extracellular TMPRSS13 stem region located between the transmembrane domain and the first site of N-linked glycosylation at asparagine (N)-250 in the scavenger receptor cysteine rich (SRCR) domain. Importantly, the catalytic competence of TMPRSS13 is essential for stem region cleavage, suggesting an autonomous mechanism of action. Site-directed mutagenesis of the 10 basic amino acids (four arginine and six lysine residues) in this region abrogated zymogen activation and catalytic activity of TMPRSS13, as well as phosphorylation, cell surface expression, and shedding. Mutation analysis of individual arginine residues identified R223, a residue located between the low-density lipoprotein receptor class A domain and the SRCR domain, as important for stem region cleavage. Mutation of R223 causes a reduction in the aforementioned functional processing steps of TMPRSS13. These data provide further insight into the roles of different post-translational modifications as regulators of the function and localization of TMPRSS13. Additionally, the data suggest the presence of complex interconnected regulatory mechanisms that may serve to ensure the proper levels of cell-surface and pericellular TMPRSS13-mediated proteolysis under homeostatic conditions.

Matriptase-2/NR4A3 axis switches TGF-β action toward suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-β action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-β1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-β/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

Modulation of Cellular Function by the Urokinase Receptor Signalling: A Mechanistic View.

Urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycosyl-phosphatidyl-inositol anchored (GPI) membrane protein. The uPAR primary ligand is the serine protease urokinase (uPA), converting plasminogen into plasmin, a broad spectrum protease, active on most extracellular matrix components. Besides uPA, the uPAR binds specifically also to the matrix protein vitronectin and, therefore, is regarded also as an adhesion receptor. Complex formation of the uPAR with diverse transmembrane proteins, including integrins, formyl peptide receptors, G protein-coupled receptors and epidermal growth factor receptor results in intracellular signalling. Thus, the uPAR is a multifunctional receptor coordinating surface-associated pericellular proteolysis and signal transduction, thereby affecting physiological and pathological mechanisms. The uPAR-initiated signalling leads to remarkable cellular effects, that include increased cell migration, adhesion, survival, proliferation and invasion. Although this is beyond the scope of this review, the uPA/uPAR system is of great interest to cancer research, as it is associated to aggressive cancers and poor patient survival. Increasing evidence links the uPA/uPAR axis to epithelial to mesenchymal transition, a highly dynamic process, by which epithelial cells can convert into a mesenchymal phenotype. Furthermore, many reports indicate that the uPAR is involved in the maintenance of the stem-like phenotype and in the differentiation process of different cell types. Moreover, the levels of anchor-less, soluble form of uPAR, respond to a variety of inflammatory stimuli, including tumorigenesis and viral infections. Finally, the role of uPAR in virus infection has received increasing attention, in view of the Covid-19 pandemics and new information is becoming available. In this review, we provide a mechanistic perspective, via the detailed examination of consolidated and recent studies on the cellular responses to the multiple uPAR activities.

The Plasminogen-Activator Plasmin System in Physiological and Pathophysiological Angiogenesis.

Angiogenesis is a process associated with the migration and proliferation of endothelial cells (EC) to form new blood vessels. It is involved in various physiological and pathophysiological conditions and is controlled by a wide range of proangiogenic and antiangiogenic molecules. The plasminogen activator–plasmin system plays a major role in the extracellular matrix remodeling process necessary for angiogenesis. Urokinase/tissue-type plasminogen activators (uPA/tPA) convert plasminogen into the active enzyme plasmin, which in turn activates matrix metalloproteinases and degrades the extracellular matrix releasing growth factors and proangiogenic molecules such as the vascular endothelial growth factor (VEGF-A). The plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of uPA and tPA, thereby an inhibitor of pericellular proteolysis and intravascular fibrinolysis, respectively. Paradoxically, PAI-1, which is expressed by EC during angiogenesis, is elevated in several cancers and is found to promote angiogenesis by regulating plasmin-mediated proteolysis and by promoting cellular migration through vitronectin. The urokinase-type plasminogen activator receptor (uPAR) also induces EC cellular migration during angiogenesis via interacting with signaling partners. Understanding the molecular functions of the plasminogen activator plasmin system and targeting angiogenesis via blocking serine proteases or their interactions with other molecules is one of the major therapeutic strategies scientists have been attracted to in controlling tumor growth and other pathological conditions characterized by neovascularization.

Abstract 3158: Characterization and targeting of TMPRSS13 in breast cancer

Abstract Breast cancer (BCa) remains a significant public health burden in the United States (US) and globally, despite advances in screening, detection, and therapeutics. In the US alone, BCa is the most commonly diagnosed cancer and the second leading cause of cancer deaths in women. The discovery of novel, targetable biomarkers is therefore paramount to reduce the morbidity and mortality associated with BCa. Cancer progression is often accompanied by increased expression of pericellular proteases capable of degrading the extracellular matrix and cleaving pro-oncogenic signaling molecules. BCa cells use pericellular proteolysis in order to enhance their proliferation, survival, and invasiveness. TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, has emerged as a potential biomarker and therapeutic target in BCa. Prior data indicates that TMPRSS13 is significantly upregulated in invasive ductal carcinoma samples at the mRNA and protein levels compared to normal breast tissue. Furthermore, TMPRSS13-deficient mice have few phenotypic defects but in a genetic model of mammary cancer, TMPRSS13-deficient mice have a significantly reduced tumor burden as well as a reduction in lung metastases. This data indicates that TMPRSS13 may be a worthwhile target to treat BCa patients. However, selective targeting of TMPRSS13 above other TTSPs is difficult due to the homology of their catalytic domains. Therefore, any information that we can glean regarding the unique biochemical and oncogenic properties of TMPRSS13 will aid in the future development of inhibitors. Our group has found that TMPRSS13 is the only TTSP family member confirmed to be phosphorylated in its intracellular domain. This phosphorylation is critical for the trafficking and cell surface expression of the protease. Mutagenesis of individual amino acids as well as deletion constructs of the phosphorylated residues in the intracellular domain have indicated that the inability of TMPRSS13 to be phosphorylated does not affect its catalytic activity, but inhibits proper cell membrane localization. Thus, phosphorylation may be imperative for TMPRSS13 to interact with pro-oncogenic substrates in the tumor microenvironment. Furthermore, preliminary data has indicated that TMPRSS13 may be a substrate for phosphorylation by CDK5, a neuronal kinase that has recently emerged as pro-oncogenic in various cancer types. CDK5, when bound to myristoylated p35, likely phosphorylates TMPRSS13 at the cell membrane, allowing TMPRSS13 to stably localize to the cell surface of both luminal and triple negative BCa cells. The overarching goal of our project is to fully understand the manner in which TMPRSS13 promotes the development and progression of BCa, which will allow for the design of selective targeted inhibitors in the future. Citation Format: Carly E. Martin, Andrew S. Murray, Evan Harrison, Karin List. Characterization and targeting of TMPRSS13 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3158.

Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma.

Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.

Let-7f miRNA regulates SDF-1\u03b1- and hypoxia-promoted migration of mesenchymal stem cells and attenuates mammary tumor growth upon exosomal release

Bone marrow-derived human mesenchymal stem cells (hMSCs) are recruited to damaged or inflamed tissues where they contribute to tissue repair. This multi-step process involves chemokine-directed invasion of hMSCs and on-site release of factors that influence target cells or tumor tissues. However, the underlying molecular mechanisms are largely unclear. Previously, we described that microRNA let-7f controls hMSC differentiation. Here, we investigated the role of let-7f in chemotactic invasion and paracrine anti-tumor effects. Incubation with stromal cell-derived factor-1α (SDF-1α) or inflammatory cytokines upregulated let-7f expression in hMSCs. Transfection of hMSCs with let-7f mimics enhanced CXCR4-dependent invasion by augmentation of pericellular proteolysis and release of matrix metalloproteinase-9. Hypoxia-induced stabilization of the hypoxia-inducible factor 1 alpha in hMSCs promoted cell invasion via let-7f and activation of autophagy. Dependent on its endogenous level, let-7f facilitated hMSC motility and invasion through regulation of the autophagic flux in these cells. In addition, secreted let-7f encapsulated in exosomes was increased upon upregulation of endogenous let-7f by treatment of the cells with SDF-1α, hypoxia, or induction of autophagy. In recipient 4T1 tumor cells, hMSC-derived exosomal let-7f attenuated proliferation and invasion. Moreover, implantation of 3D spheroids composed of hMSCs and 4T1 cells into a breast cancer mouse model demonstrated that hMSCs overexpressing let-7f inhibited tumor growth in vivo. Our findings provide evidence that let-7f is pivotal in the regulation of hMSC invasion in response to inflammation and hypoxia, suggesting that exosomal let-7f exhibits paracrine anti-tumor effects.

Tissue remodeling by invadosomes.

One of the strategies used by cells to degrade and remodel the extracellular matrix (ECM) is based on invadosomes, actin-based force-producing cell–ECM contacts that function in adhesion and migration and are characterized by their capacity to mediate pericellular proteolysis of ECM components. Invadosomes found in normal cells are called podosomes, whereas invadosomes of invading cancer cells are named invadopodia. Despite their broad involvement in cell migration and in protease-dependent ECM remodeling and their detection in living organisms and in fresh tumor tissue specimens, the specific composition and dynamic behavior of podosomes and invadopodia and their functional relevance in vivo remain poorly understood. Here, we discuss recent findings that underline commonalities and peculiarities of podosome and invadopodia in terms of organization and function and propose an updated definition of these cellular protrusions, which are increasingly relevant in patho-physiological tissue remodeling.

A Narrative Review on Plasminogen Activator Inhibitor-1 and Its (Patho)Physiological Role: To Target or Not to Target?

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of plasminogen activators (PAs) and is therefore an important inhibitor of the plasminogen/plasmin system. Being the fast-acting inhibitor of tissue-type PA (tPA), PAI-1 primarily attenuates fibrinolysis. Through inhibition of urokinase-type PA (uPA) and interaction with biological ligands such as vitronectin and cell-surface receptors, the function of PAI-1 extends to pericellular proteolysis, tissue remodeling and other processes including cell migration. This review aims at providing a general overview of the properties of PAI-1 and the role it plays in many biological processes and touches upon the possible use of PAI-1 inhibitors as therapeutics.

Surface-bound matrix metalloproteinase-8 on macrophages: Contributions to macrophage pericellular proteolysis and migration through tissue barriers.

ABSTRACTObjectiveMMP‐8 binds to surface‐bound tissue inhibitor of metalloproteinase‐1 (TIMP‐1) on PMNs to promote pericellular proteolysis during the development of inflammatory diseases associated with tissue destruction. Little is known about the biology of MMP‐8 in macrophages. We tested the hypotheses that: (1) MMP‐8 and TIMP‐1 are also expressed on the surface of activated macrophages, (2) surface‐bound MMP‐8 on macrophages promotes TIMP‐resistant pericellular proteolysis and macrophage migration through tissue barriers, and (3) MMP‐8 binds to surface‐bound TIMP‐1 on macrophages.MethodsSurface MMP‐8 and TIMP‐1 levels were measured on human monocyte‐derived macrophages (MDM) and/or murine macrophages using immunostaining, biotin‐labeling, and substrate cleavage methods. The susceptibility of membrane‐bound Mmp‐8 on activated macrophages from wild‐type (WT) mice to TIMPs was measured. Migration of WT and Mmp‐8 −/− macrophages through models of tissue barriers in vitro and the accumulation of peritoneal macrophages in WT versus Mmp‐8 −/− mice with sterile peritonitis was compared. Surface levels of Mmp‐8 were compared on activated macrophages from WT and Timp‐1 −/− mice.ResultsLipopolysaccharides and a cluster of differentiation 40 ligand increased surface MMP‐8 and/or TIMP‐1 staining and surface type I collagenase activity on MDM and/or murine macrophages. Activated Mmp‐8 −/− macrophages degraded less type I collagen than activated WT macrophages. The surface type‐I collagenase activity on WT macrophages was resistant to inhibition by Timp‐1. Peritoneal macrophage accumulation was similar in WT and Mmp‐8 −/− mice with sterile acute peritonitis. However, Mmp‐8 −/− macrophages migrated less efficiently through models of tissue barriers (especially those containing type I collagen) than WT cells. Activated WT and Timp‐1 −/− macrophages had similar surface‐bound Mmp‐8 levels.ConclusionsMMP‐8 and TIMP‐1 are expressed on the surface of activated human MDM and murine macrophages, but Mmp‐8 is unlikely to bind to surface‐bound Timp‐1 on these cells. Surface‐bound MMP‐8 contributes to TIMP‐resistant monocyte/macrophage pericellular proteolysis and macrophage migration through collagen‐containing tissue barriers.

Lack of Plasminogen Activator Inhibitor-1 Effect in a Transgenic Mouse Model of Metastatic Melanoma

Tumor cell invasion and metastasis is a complex, multistep process that is postulated to require degradation of extracellular matrix at several steps. Urokinase-type plasminogen activator (uPA) is expressed on the cell surface of B16 murine melanoma cells and is thought to contribute to the pericellular proteolysis necessary for tumor cell migration. In vitro modification of B16 melanoma cell surface uPA activity has been shown to alter the invasive and metastatic potential of these murine melanoma cells in vivo. Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of both uPA and tissue-type plasminogen activator (tPA) is the major physiologic regulator of plasminogen activator activity. To test the role of host PAI-1 in the invasive and metastatic capacity of B16 melanoma cells we analyzed local tumor growth and pulmonary metastasis in transgenic mice engineered to overexpress murine PAI-1 in multiple tissues including lung, and in mice completely deficient in PAI-1. No significant difference in the number of pulmonary metastases was observed after intravenous inoculation of tumor cells into PAI-1-overexpressing and PAI-1-deficient mice when compared with wild-type controls. Similarly, in a spontaneous metastasis model, PAI-1-overexpressing and PAI-1-deficient mice demonstrated no difference in primary tumor size or overall survival. These data demonstrate that wide variations of host PAI-1 expression, from complete absence to marked overexpression, does not significantly influence the metastatic potential of B16 melanoma cells in a murine model.

Specific inhibitors of urokinase plasminogen activator for treatment of cancers; In silico approach

Invasion, metastasis and angiogenesis are fundamental processes in the development of solid cancers. All these depend on the proteolysis where plasminogen activation system (PAS) is the prominent component. Plasmin of PAS, with a broad spectrum of proteins lysed, is secreted as a pro-enzyme and then activated by urokinase (uPA) or tissue plasminogen activator (tPA). Urokinase is in control of pericellular proteolysis while tPA mediates intravascular fibrinolysis. The most effective way to reduce excessive activity of plasmin in cancers is by inactivation of its activators (PAs). Inhibition of PAs reduces tumor size of cancers in vivo. However, for successful targeted anticancer therapy it is essential to find specific uPA inhibitors and to protect normal function of tPA. Unfortunately, most known inhibitors are unspecific, acting on both PAs. PAs are highly homologous enzymes with extreme similarities in their active sites so large numbers of chemicals need to be tested to find novel specific uPA inhibitors. Methods: As the availability of 3D protein structures determined experimentally by X-ray crystallography is growing, computational methods are ever more used in targeted drug discovery. AutoDock Vina molecular docking, exploring binding of small molecules to target protein using a Monte Carlo technique and scoring function, is gaining popularity due to its excellent prediction accuracy. Results: Using AutoDock we have found many inhibitors of PA from our 3D database of 6170 compounds which bind in silico to X-ray structures of the specificity pocket (B187-B197, B212-B229) of both PAs preventing activation of plasminogen. However we were successful in identifying a few molecules which are specific for uPA. For example: both amiloride and chrysin bind to the specificity pocket of uPA but not to tPA. We have found that they bind to other parts of tPA, distant from the specificity pocket, thus preserving the tPA enzymatic activity while being effective toward uPA. Conclusion: In silico search yield specific inhibitors of uPA which, when verified for safety and efficiency by in vivo testing, could be used as novel therapeutics to limit metastasis and angiogenesis in anticancer therapy.

Characterization of an Extensive Interface on Vitronectin for Binding to Plasminogen Activator Inhibitor-1: Adoption of Structure in an Intrinsically Disordered Region.

Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell adhesion/migration and proteolytic cascades that govern hemostasis and pericellular proteolysis. Many of these functions occur via interactions with its binding partner, plasminogen activator inhibitor-1 (PAI-1), the chief inhibitor of proteases that lyse and activate plasminogen. We focused on a region of vitronectin that remains uncharacterized from previous X-ray scattering, nuclear magnetic resonance, and computational modeling approaches and which we propose is involved in binding to PAI-1. This region, which bridges the N-terminal somatomedin B (SMB) domain with a large central β-propeller domain of vitronectin, appears unstructured and has characteristics of an intrinsically disordered domain (IDD). The effect of osmolytes was evaluated using circular dichroism and SANS to explore the potential of the IDD to undergo a disorder-to-order transition. The results suggest that the IDD favors a more ordered structure under osmotic pressure; SANS shows a smaller radius of gyration (Rg) and a more compact fold of the IDD upon addition of osmolytes. To test whether PAI-1 binding is also coupled to folding within the IDD structure, a set of SANS experiments with contrast variation were performed on the complex of PAI-1 with a vitronectin fragment corresponding to the N-terminal 130 amino acids (denoted the SMB-IDD because it contains the SMB domain and IDD in linear sequence). Analysis of the SANS data using the Ensemble Optimization Method confirms that the SMB-IDD adopts a more compact configuration when bound to PAI-1. Calculated structures for the PAI-1:SMB-IDD complex suggest that the IDD provides an interaction surface outside of the primary PAI-1-binding site located within the SMB domain; this binding is proposed to lead to the assembly of higher-order structures of vitronectin and PAI-1 commonly found in tissues.

Nerve, Muscle, and Synaptogenesis.

The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function, and development. Over the last several decades, a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the current model also proposes that agrin can serve two functions, to induce synaptogenesis and to stabilize new synapses, once these are formed. This review examines the evidence for these propositions, and concludes that it remains possible that a single molecular mechanism mediates synaptogenesis at all NMJs, and that agrin acts as a stabilizer, while its role as inducer is open to question. Moreover, if agrin does not act to initiate synaptogenesis, it follows that as yet uncharacterized molecular interactions are required to play this essential inductive role. Several alternatives to agrin for this function are suggested, including focal pericellular proteolysis and integrin signaling, but all require experimental validation.

Tissue Inhibitor of Metalloproteinase-1 Promotes Polymorphonuclear Neutrophil (PMN) Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces.

Matrix metalloproteinase (MMP)-8 and -9 released by degranulating polymorphonuclear cells (PMNs) promote pericellular proteolysis by binding to PMN surfaces in a catalytically active tissue inhibitor of metalloproteinases (TIMP)-resistant forms. The PMN receptor(s) to which MMP-8 and MMP-9 bind(s) is not known. Competitive binding experiments showed that Mmp-8 and Mmp-9 share binding sites on murine PMN surfaces. A novel form of TIMP-1 (an inhibitor of soluble MMPs) is rapidly expressed on PMN surfaces when human PMNs are activated. Membrane-bound TIMP-1 is the PMN receptor for pro- and active MMP-8 and -9 as shown by the following: 1) TIMP-1 is strikingly colocalized with MMP-8 and -9 on activated human PMN surfaces and in PMN extracellular traps; 2) minimal immunoreactive and active Mmp-8 or Mmp-9 are detected on the surface of activated Timp-1-/- murine PMNs; and 3) binding of exogenous Timp-1 (but not Timp-2) to Timp-1-/- murine PMNs reconstitutes the binding of exogenous pro-Mmp-8 and pro-Mmp-9 to the surface of Timp-1-/- PMNs. Unlike full-length pro-Mmp-8 and pro-Mmp-9, mutant pro-Mmp proteins lacking the COOH-terminal hemopexin domain fail to bind to Mmp-8-/-x Mmp-9-/- murine PMNs. Soluble hemopexin inhibits the binding of pro-Mmp-8 and pro-Mmp-9 to Mmp-8-/-x Mmp-9-/- murine PMNs. Thus, the COOH-terminal hemopexin domains of pro-Mmp-8 and pro-Mmp-9 are required for their binding to membrane-bound Timp-1 on murine PMNs. Exposing nonhuman primates to cigarette smoke upregulates colocalized expression of TIMP-1 with MMP-8 and MMP-9 on peripheral blood PMN surfaces. By anchoring MMP-8 and MMP-9 to PMN surfaces, membrane-bound TIMP-1 plays a counterintuitive role in promoting PMN pericellular proteolysis occurring in chronic obstructive pulmonary disease and other diseases.