Identification of Annexin A4 as a hepatopancreas factor involved in liver cell survival.

To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing ß-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic ß-cell apoptosis and cancer cell chemoresistance.

Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.

It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and γ-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differentially regulate cell motility in a two-dimensional versus three-dimensional environment. We also demonstrated that in polarized cancer cells, the levels of PTK7 expression and proteolysis were directly linked to the structure and kinetics of cell protrusions, including lamellipodia and invadopodia. In the functionally relevant and widely accepted animal models of metastasis, mouse and chick embryo models, both the overexpression and knock-out of PTK7 in HT1080 cells abrogated metastatic dissemination. Our analysis of human tissue specimens confirmed intensive proteolysis of PTK7 in colorectal cancer tumors, but not in matching normal tissue. Our results provide convincing evidence that both PTK7 expression and proteolysis, rather than the level of the cellular full-length PTK7 alone, contribute to efficient directional cell motility and metastasis in cancer.

Downstream signaling and genome-wide regulatory effects of PTK7 pseudokinase and its proteolytic fragments in cancer cells.

BACKGROUND: The full-length membrane protein tyrosine kinase 7 (PTK7) pseudokinase, an important component of the planar cell polarity and the Wnt canonical and non-canonical pathways, is a subject of step-wise proteolysis in cells and tissues. The proteolysis of PTK7 involves membrane type-matrix metalloproteinase (MT1-MMP), members of the Disintegrin Domain and Metalloproteinase (ADAM) family, and γ-secretase. This multi-step proteolysis results in the generation of the digest fragments of PTK7. These fragments may be either liberated into the extracellular milieu or retained on the plasma membrane or released into the cytoplasm and then transported into the nucleus. RESULTS: We employed the genome-wide transcriptional and kinome array analyses to determine the role of the full-length membrane PTK7 and its proteolytic fragments in the downstream regulatory mechanisms, with an emphasis on the cell migration-related genes and proteins. Using fibrosarcoma HT1080 cells stably expressing PTK7 and its mutant and truncated species, the structure of which corresponded to the major PTK7 digest fragments, we demonstrated that the full-length membrane 1-1070 PTK7, the N-terminal 1-694 soluble ectodomain fragment, and the C-terminal 622-1070 and 726-1070 fragments differentially regulate multiple genes and signaling pathways in our highly invasive cancer cell model. Immunoblotting of the selected proteins were used to validate the results of our high throughput assays. CONCLUSIONS: Our results suggest that PTK7 levels need to be tightly controlled to enable migration and that the anti-migratory effect of the full-length membrane PTK7 is linked to the down-regulation of multiple migration-related genes and to the activation of the Akt and c-Jun pathway. In turn, the C-terminal fragments of PTK7 act predominantly via the RAS-ERK and CREB/ATF1 pathway and through the up-regulation of cadherin-11. In general, our data correlate well with the distinct functionality of the full-length receptor tyrosine kinases and their respective intracellular domain (ICD) proteolytic fragments.

Substrate cleavage profiling suggests a distinct function of Bacteroides fragilis metalloproteinases (fragilysin and metalloproteinase II) at the microbiome-inflammation-cancer interface.

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Non-destructive and selective imaging of the functionally active, pro-invasive membrane type-1 matrix metalloproteinase (MT1-MMP) enzyme in cancer cells.

Proteolytic activity of cell surface-associated MT1-matrix metalloproteinase (MMP) (MMP-14) is directly related to cell migration, invasion, and metastasis. MT1-MMP is regulated as a proteinase by activation and conversion of the latent proenzyme into the active enzyme, and also via inhibition by tissue inhibitors of MMPs (TIMPs) and self-proteolysis. MT1-MMP is also regulated as a membrane protein through its internalization and recycling. Routine immunohistochemistry, flow cytometry, reverse transcription-PCR, and immunoblotting methodologies do not allow quantitative imaging and assessment of the cell-surface levels of the active, TIMP-free MT1-MMP enzyme. Here, we developed a fluorescent reporter prototype that targets the cellular active MT1-MMP enzyme alone. The reporter (MP-3653) represents a liposome tagged with a fluorochrome and functionalized with a PEG chain spacer linked to an inhibitory hydroxamate warhead. Our studies using the MP-3653 reporter and its inactive derivative demonstrated that MP-3653 can be efficiently used not only to visualize the trafficking of MT1-MMP through the cell compartment, but also to quantify the femtomolar range amounts of the cell surface-associated active MT1-MMP enzyme in multiple cancer cell types, including breast carcinoma, fibrosarcoma, and melanoma. Thus, the levels of the naturally expressed, fully functional, active cellular MT1-MMP enzyme are roughly equal to 1 × 10(5) molecules/cell, whereas these levels are in a 1 × 10(6) range in the cells with the enforced MT1-MMP expression. We suggest that the reporter we developed will contribute to the laboratory studies of MT1-MMP and then, ultimately, to the design of novel, more efficient prognostic approaches and personalized cancer therapies.

High-resolution analysis and functional mapping of cleavage sites and substrate proteins of furin in the human proteome.

BACKGROUND: There is a growing appreciation of the role of proteolytic processes in human health and disease, but tools for analysis of such processes on a proteome-wide scale are limited. Furin is a ubiquitous proprotein convertase that cleaves after basic residues and transforms secretory proproteins into biologically active proteins. Despite this important role, many furin substrates remain unknown in the human proteome. METHODOLOGY/PRINCIPAL FINDINGS: We devised an approach for proteinase target identification that combines an in silico discovery pipeline with highly multiplexed proteinase activity assays. We performed in silico analysis of the human proteome and identified over 1,050 secretory proteins as potential furin substrates. We then used a multiplexed protease assay to validate these tentative targets. The assay was carried out on over 3,260 overlapping peptides designed to represent P7-P1' and P4-P4' positions of furin cleavage sites in the candidate proteins. The obtained results greatly increased our knowledge of the unique cleavage preferences of furin, revealed the importance of both short-range (P4-P1) and long-range (P7-P6) interactions in defining furin cleavage specificity, demonstrated that the R-X-R/K/X-R ↓ motif alone is insufficient for predicting furin proteolysis of the substrate, and identified ≈ 490 potential protein substrates of furin in the human proteome. CONCLUSIONS/SIGNIFICANCE: The assignment of these substrates to cellular pathways suggests an important role of furin in development, including axonal guidance, cardiogenesis, and maintenance of stem cell pluripotency. The novel approach proposed in this study can be readily applied to other proteinases.

Insights into ectodomain shedding and processing of protein-tyrosine pseudokinase 7 (PTK7).

The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP(621)↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.

Immunodominant fragments of myelin basic protein initiate T cell-dependent pain.

BACKGROUND: The myelin sheath provides electrical insulation of mechanosensory Aß-afferent fibers. Myelin-degrading matrix metalloproteinases (MMPs) damage the myelin sheath. The resulting electrical instability of Aß-fibers is believed to activate the nociceptive circuitry in Aß-fibers and initiate pain from innocuous tactile stimulation (mechanical allodynia). The precise molecular mechanisms, responsible for the development of this neuropathic pain state after nerve injury (for example, chronic constriction injury, CCI), are not well understood. METHODS AND RESULTS: Using mass spectrometry of the whole sciatic nerve proteome followed by bioinformatics analyses, we determined that the pathways, which are classified as the Infectious Disease and T-helper cell signaling, are readily activated in the nerves post-CCI. Inhibition of MMP-9/MMP-2 suppressed CCI-induced mechanical allodynia and concomitant TNF-α and IL-17A expression in nerves. MMP-9 proteolysis of myelin basic protein (MBP) generated the MBP84-104 and MBP68-86 digest peptides, which are prominent immunogenic epitopes. In agreement, the endogenous MBP69-86 epitope co-localized with MHCII and MMP-9 in Schwann cells and along the nodes of Ranvier. Administration of either the MBP84-104 or MBP68-86 peptides into the naïve nerve rapidly produced robust mechanical allodynia with a concomitant increase in T cells and MHCII-reactive cell populations at the injection site. As shown by the genome-wide expression profiling, a single intraneural MBP84-104 injection stimulated the inflammatory, immune cell trafficking, and antigen presentation pathways in the injected naïve nerves and the associated spinal cords. Both MBP84-104-induced mechanical allodynia and characteristic pathway activation were remarkably less prominent in the T cell-deficient athymic nude rats. CONCLUSIONS: These data implicate MBP as a novel mediator of pain. Furthermore, the action of MMPs expressed within 1 day post-injury is critical to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state.

The MMP-9/TIMP-1 axis controls the status of differentiation and function of myelin-forming Schwann cells in nerve regeneration.

BACKGROUND: Myelinating Schwann cells (mSCs) form myelin in the peripheral nervous system. Because of the works by us and others, matrix metalloproteinase-9 (MMP-9) has recently emerged as an essential component of the Schwann cell signaling network during sciatic nerve regeneration. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using the genome-wide transcriptional profiling of normal and injured sciatic nerves in mice followed by extensive bioinformatics analyses of the data, we determined that an endogenous, specific MMP-9 inhibitor [tissue inhibitor of metalloproteinases (TIMP)-1] was a top up-regulated gene in the injured nerve. MMP-9 capture followed by gelatin zymography and Western blotting of the isolated samples revealed the presence of the MMP-9/TIMP-1 heterodimers and the activated MMP-9 enzyme in the injured nerve within the first 24 h post-injury. MMP-9 and TIMP-1 co-localized in mSCs. Knockout of the MMP-9 gene in mice resulted in elevated numbers of de-differentiated/immature mSCs in the damaged nerve. Our comparative studies using MMP-9 knockout and wild-type mice documented an aberrantly enhanced proliferative activity and, accordingly, an increased number of post-mitotic Schwann cells, short internodes and additional nodal abnormalities in remyelinated nerves of MMP-9 knockout mice. These data imply that during the first days post-injury MMP-9 exhibits a functionally important anti-mitogenic activity in the wild-type mice. Pharmacological inhibition of MMP activity suppressed the expression of Na(v)1.7/1.8 channels in the crushed nerves. CONCLUSION/SIGNIFICANCE: Collectively, our data established an essential role of the MMP-9/TIMP-1 axis in guiding the mSC differentiation and the molecular assembly of myelin domains in the course of the nerve repair process. Our findings of the MMP-dependent regulation of Na(v) channels, which we document here for the first time, provide a basis for therapeutic intervention in sensorimotor pathologies and pain.

Novel MT1-MMP small-molecule inhibitors based on insights into hemopexin domain function in tumor growth.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a promising drug target in malignancy. The structure of MT1-MMP includes the hemopexin domain (PEX) that is distinct from and additional to the catalytic domain. Current MMP inhibitors target the conserved active site in the catalytic domain and, as a result, repress the proteolytic activity of multiple MMPs instead of MT1-MMP alone. In our search for noncatalytic inhibitors of MT1-MMP, we compared the protumorigenic activity of wild-type MT1-MMP with an MT1-MMP mutant lacking PEX (ΔPEX). In contrast to MT1-MMP, ΔPEX did not support tumor growth in vivo, and its expression resulted in small fibrotic tumors that contained increased levels of collagen. Because these findings suggested an important role for PEX in tumor growth, we carried out an inhibitor screen to identify small molecules targeting the PEX domain of MT1-MMP. Using the Developmental Therapeutics Program (National Cancer Institute/NIH), virtual ligand screening compound library as a source and the X-ray crystal structure of PEX as a target, we identified and validated a novel PEX inhibitor. Low dosage, intratumoral injections of PEX inhibitor repressed tumor growth and caused a fibrotic, ΔPEX-like tumor phenotype in vivo. Together, our findings provide a preclinical proof of principle rationale for the development of novel and selective MT1-MMP inhibitors that specifically target the PEX domain.

Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity.

Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-ß (Aß) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aß-peptide deposits [composed mainly of wild-type (WT) Aß(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aß(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aß(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aß(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aß(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.

Intradomain cleavage of inhibitory prodomain is essential to protumorigenic function of membrane type-1 matrix metalloproteinase (MT1-MMP) in vivo.

Invasive cancers use pericellular proteolysis to breach the extracellular matrix and basement membrane barriers and invade the surrounding tissue. Proinvasive membrane type-1 matrix metalloproteinase (MT1-MMP) is the primary mediator of proteolytic events on the cancer cell surface. MT1-MMP is synthesized as a zymogen. The latency of MT1-MMP is maintained by its N-terminal inhibitory prodomain. In the course of MT1-MMP activation, the R(108)RKR(111) ↓ Y(112) prodomain sequence is processed by furin. The intact prodomain released by furin alone, however, is a potent inhibitor of the emerging MT1-MMP enzyme. Evidence suggests that the prodomain undergoes intradomain cleavage at the PGD ↓ L(50) site followed by the release of the degraded prodomain by furin cleavage that finalizes the two-step activation event. These cleavages, only if combined, cause the activation of MT1-MMP. The significance of the intradomain cleavage in the protumorigenic program of MT1-MMP, however, remained unidentified. To identify this important parameter, in our current study, we used the cells that expressed the wild-type prodomain-based fluorescent biosensor and the mutant biosensor with the inactivated PGD↓L(50) cleavage site (L50D mutant) and also the cells with the enforced expression of the wild-type and L50D mutant MT1-MMP. Using cell-based tests, orthotopic breast cancer xenografts in mice, and genome-wide transcriptional profiling of cultured cells and tumor xenografts, we demonstrated that the intradomain cleavage of the PGD ↓ L(50) sequence of the prodomain is essential for the protumorigenic function of MT1-MMP. Our results emphasize the importance of the intradomain cleavages resulting in the inactivation of the respective inhibitory prodomains not only for MT1-MMP but also for other MMP family members.

Potential relation of aberrant proteolysis of human protein tyrosine kinase 7 (PTK7) chuzhoi by membrane type 1 matrix metalloproteinase (MT1-MMP) to congenital defects.

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.

The Wnt/planar cell polarity protein-tyrosine kinase-7 (PTK7) is a highly efficient proteolytic target of membrane type-1 matrix metalloproteinase: implications in cancer and embryogenesis.

PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.

Mass spectrometry of murine leukemia virus core proteins.

A mass spectrometry (MS) approach was used to analyze viral core proteins of the murine leukemia virus (MuLV)-based gene delivery vector. The retroviral particles produced by traditional methods were concentrated and purified by ultracentrifugation and spin column for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS. MALDI application detected all core MuLV proteins, partial degradation of p10, phosphorylation of p12, as well as the previously unknown formation of a polymeric supramolecular complex between p15 and p30 core proteins. ESI provided information on the post-translational modifications of MuLV core proteins. Data suggest myristoylation of p15 and oxidation of methionine residues in both p12 and p30, whereas cysteine residues in p10, p15 and p30 were not oxidized. The current study demonstrates that MALDI and ESI are efficient tools for viral core protein analysis and can be used as analytical tools in virology and biotechnology of gene delivery vectors.

Internal cleavages of the autoinhibitory prodomain are required for membrane type 1 matrix metalloproteinase activation, although furin cleavage alone generates inactive proteinase.

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.

A femtomol range FRET biosensor reports exceedingly low levels of cell surface furin: implications for the processing of anthrax protective antigen.

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR / STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.

Microarray-based transcriptional and epigenetic profiling of matrix metalloproteinases, collagens, and related genes in cancer.

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Selective and potent furin inhibitors protect cells from anthrax without significant toxicity.

Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.

Timp-2 binding with cellular MT1-MMP stimulates invasion-promoting MEK/ERK signaling in cancer cells.

Both invasion-promoting MT1-MMP and its physiological inhibitor TIMP-2 play a significant role in tumorigenesis and are identified in the most aggressive cancers. Despite its antiproteolytic effects in vitro, clinical data suggest that TIMP-2 expression is positively associated with tumor recurrence, thus emphasizing the wide-ranging role of TIMP-2 in malignancies. To shed light on this role of TIMP-2, we report that low concentrations of TIMP-2, by interacting with MT1-MMP (a specific membrane receptor of TIMP-2), induce the MEK/ERK signaling cascade in fibrosarcoma HT1080 cells which express MT1-MMP naturally. TIMP-2 binding with cell surface-associated MT1-MMP stimulates phosphorylation of MEK1/2, which is upstream of ERK1/2, and the ERK1/2 substrate p90RSK. Consistent with volumes of literature, we confirmed that the activation of ERK stimulated cell migration. Both the transcriptional silencing of MT1-MMP and the inhibition of MEK1/2 reversed the signaling effects of TIMP-2/MT1-MMP while the active site-targeting MMP inhibitor GM6001 did not. Our data suggest that both the interactions of TIMP-2 with MT1-MMP, which activate the pro-migratory ERK signaling cascade,and the conventional inhibition of MT1-MMP's catalytic activity by TIMP-2, play a role in the invasion-promoting function of MT1-MMP. The TIMP-2-induced stimulation of ERK signaling in cancer cells explains the direct, as opposed to the inverse, association of TIMP-2 expression with poor prognosis in cancer.