A myelin basic protein fragment induces sexually dimorphic transcriptome signatures of neuropathic pain in mice.

In the peripheral nerve, mechanosensitive axons are insulated by myelin, a multilamellar membrane formed by Schwann cells. Here, we offer first evidence that a myelin degradation product induces mechanical hypersensitivity and global transcriptomics changes in a sex-specific manner. Focusing on downstream signaling events of the functionally active 84-104 myelin basic protein (MBP(84-104)) fragment released after nerve injury, we demonstrate that exposing the sciatic nerve to MBP(84-104) via endoneurial injection produces robust mechanical hypersensitivity in female, but not in male, mice. RNA-seq and systems biology analysis revealed a striking sexual dimorphism in molecular signatures of the dorsal root ganglia (DRG) and spinal cord response, not observed at the nerve injection site. Mechanistically, intra-sciatic MBP(84-104) induced phospholipase C (PLC)-driven (females) and phosphoinositide 3-kinase-driven (males) phospholipid metabolism (tier 1). PLC/inositol trisphosphate receptor (IP3R) and estrogen receptor co-regulation in spinal cord yielded Ca2+-dependent nociceptive signaling induction in females that was suppressed in males (tier 2). IP3R inactivation by intrathecal xestospongin C attenuated the female-specific hypersensitivity induced by MBP(84-104). According to sustained sensitization in tiers 1 and 2, T cell-related signaling spreads to the DRG and spinal cord in females, but remains localized to the sciatic nerve in males (tier 3). These results are consistent with our previous finding that MBP(84-104)-induced pain is T cell-dependent. In summary, an autoantigenic peptide endogenously released in nerve injury triggers multisite, sex-specific transcriptome changes, leading to neuropathic pain only in female mice. MBP(84-104) acts through sustained co-activation of metabolic, estrogen receptor-mediated nociceptive, and autoimmune signaling programs.

Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.

Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.

Interaction of the cryptic fragment of myelin basic protein with mitochondrial voltage-dependent anion-selective channel-1 affects cell energy metabolism.

In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84-104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down assay, mass spectrometry and molecular modeling studies suggested that, similar with many other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anion-selective channel-1 (VDAC-1), a mitochondrial porin. Molecular modeling suggested that MBP84-104 directly binds to the N-terminal α-helix located midway inside the 19 ß-blade barrel of VDAC-1. These interactions may have affected the mitochondrial functions and energy metabolism in multiple cell types. Notably, MBP84-104 caused neither cell apoptosis nor affected the total cellular ATP levels, but repressed the aerobic glycolysis (lactic acid fermentation) and decreased the l-lactate/d-glucose ratio (also termed as the Warburg effect) in normal and cancer cells. Overall, our findings implied that because of its interactions with VDAC-1, the cryptic MBP84-104 peptide invoked reprogramming of the cellular energy metabolism that favored enhanced cellular activity, rather than apoptotic cell death. We concluded that the released MBP84-104 peptide, internalized by the cells, contributes to the reprogramming of the energy-generating pathways in multiple cell types.

Active-site MMP-selective antibody inhibitors discovered from convex paratope synthetic libraries.

Proteases are frequent pharmacological targets, and their inhibitors are valuable drugs in multiple pathologies. The catalytic mechanism and the active-site fold, however, are largely conserved among the protease classes, making the development of the selective inhibitors exceedingly challenging. In our departure from the conventional strategies, we reviewed the structure of known camelid inhibitory antibodies, which block enzyme activities via their unusually long, convex-shaped paratopes. We synthesized the human Fab antibody library (over 1.25 × 109 individual variants) that carried the extended, 23- to 27-residue, complementarity-determining region (CDR)-H3 segments. As a proof of principle, we used the catalytic domain of matrix metalloproteinase-14 (MMP-14), a promalignant protease and a drug target in cancer, as bait. In our screens, we identified 20 binders, of which 14 performed as potent and selective inhibitors of MMP-14 rather than as broad-specificity antagonists. Specifically, Fab 3A2 bound to MMP-14 in the vicinity of the active pocket with a high 4.8 nM affinity and was similarly efficient (9.7 nM) in inhibiting the protease cleavage activity. We suggest that the convex paratope antibody libraries described here could be readily generalized to facilitate the design of the antibody inhibitors to many additional enzymes.

Matrix metalloproteinases - From the cleavage data to the prediction tools and beyond.

Understanding the physiological role of any protease requires identification of both its cleavage substrates and their relative cleavage efficacy as compared with other substrates and other proteinases. Our review manuscript is focused on the cleavage preferences of the individual matrix metalloproteinases (MMPs) and the cleavage similarity and distinction that exist in the human MMP family. The recent in-depth analysis of MMPs by us and many others greatly increased knowledge of the MMP biology and structural-functional relationships among this protease family members. A better knowledge of cleavage preferences of MMPs has led us to the development of the prediction tools that are now capable of the high throughput reliable prediction and ranking the MMP cleavage sites in the peptide sequences in silico. Our software unifies and consolidates volumes of the pre-existing data. Now this prediction-ranking in silico tool is ready to be used by others. The software we developed may facilitate both the identification of the novel proteolytic regulatory pathways and the discovery of the previously uncharacterized substrates of the individual MMPs. Because now the MMP research may be based on the mathematical probability parameters rather than on either random luck or common sense alone, the researchers armed with this novel in silico tool will be better equipped to fine-tune or, at least, to sharply focus their wet chemistry experiments. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Structural homology of myelin basic protein and muscarinic acetylcholine receptor: Significance in the pathogenesis of complex regional pain syndrome.

Complex regional pain syndrome is an extremely painful condition that develops after trauma to a limb. Complex regional pain syndrome exhibits autoimmune features in part mediated by autoantibodies against muscarinic-2 acetylcholine (M2) receptor. The mechanisms underlying the M2 receptor involvement in complex regional pain syndrome remain obscure. Based on our recent work demonstrating that limb nerve trauma releases a potent proalgesic, immunodominant myelin basic protein fragment, our present sequence database analyses reveal an unexpected and previously undescribed structural homology of the proalgesic myelin basic protein fragment with the M2 receptor. As both complex regional pain syndrome and the proalgesic myelin basic protein activity are prevalent in females, this myelin basic protein/M2 homology presents an inviting hypothesis explaining the mechanisms of autoimmune pathogenesis and sexual dimorphism that underlies vulnerability toward developing complex regional pain syndrome and other pain states with neuropathic features. This hypothesis may aid in the development of novel diagnostic and therapeutic strategies to chronic pain.

Acute- and late-phase matrix metalloproteinase (MMP)-9 activity is comparable in female and male rats after peripheral nerve injury.

BACKGROUND: In the peripheral nerve, pro-inflammatory matrix metalloproteinase (MMP)-9 performs essential functions in the acute response to injury. Whether MMP-9 activity contributes to late-phase injury or whether MMP-9 expression or activity after nerve injury is sexually dimorphic remains unknown. METHODS: Patterns of MMP-9 expression, activity and excretion were assessed in a model of painful peripheral neuropathy, sciatic nerve chronic constriction injury (CCI), in female and male rats. Real-time Taqman RT-PCR for MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) of nerve samples over a 2-month time course of CCI was followed by gelatin zymography of crude nerve extracts and purified MMP-9 from the extracts using gelatin Sepharose-beads. MMP excretion was determined using protease activity assay of urine in female and male rats with CCI. RESULTS: The initial upsurge in nerve MMP-9 expression at day 1 post-CCI was superseded more than 100-fold at day 28 post-CCI. The high level of MMP-9 expression in late-phase nerve injury was accompanied by the reduction in TIMP-1 level. The absence of MMP-9 in the normal nerve and the presence of multiple MMP-9 species (the proenzyme, mature enzyme, homodimers, and heterodimers) was observed at day 1 and day 28 post-CCI. The MMP-9 proenzyme and mature enzyme species dominated in the early- and late-phase nerve injury, consistent with the high and low level of TIMP-1 expression, respectively. The elevated nerve MMP-9 levels corresponded to the elevated urinary MMP excretion post-CCI. All of these findings were comparable in female and male rodents. CONCLUSION: The present study offers the first evidence for the excessive, uninhibited proteolytic MMP-9 activity during late-phase painful peripheral neuropathy and suggests that the pattern of MMP-9 expression, activity, and excretion after peripheral nerve injury is universal in both sexes.

Reciprocal relationship between membrane type 1 matrix metalloproteinase and the algesic peptides of myelin basic protein contributes to chronic neuropathic pain.

Myelin basic protein (MBP) is an auto-antigen able to induce intractable pain from innocuous mechanical stimulation (mechanical allodynia). The mechanisms provoking this algesic MBP activity remain obscure. Our present study demonstrates that membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) releases the algesic MBP peptides from the damaged myelin, which then reciprocally enhance the expression of MT1-MMP in nerve to sustain a state of allodynia. Specifically, MT1-MMP expression and activity in rat sciatic nerve gradually increased starting at day 3 after chronic constriction injury (CCI). Inhibition of the MT1-MMP activity by intraneural injection of the function-blocking human DX2400 monoclonal antibody at day 3 post-CCI reduced mechanical allodynia and neuropathological signs of Wallerian degeneration, including axon demyelination, degeneration, edema and formation of myelin ovoids. Consistent with its role in allodynia, the MT1-MMP proteolysis of MBP generated the MBP69-86-containing epitope sequences in vitro. In agreement, the DX2400 therapy reduced the release of the MBP69-86 epitope in CCI nerve. Finally, intraneural injection of the algesic MBP69-86 and control MBP2-18 peptides differentially induced MT1-MMP and MMP-2 expression in the nerve. With these data we offer a novel, self-sustaining mechanism of persistent allodynia via the positive feedback loop between MT1-MMP and the algesic MBP peptides. Accordingly, short-term inhibition of MT1-MMP activity presents a feasible pharmacological approach to intervene in this molecular circuit and the development of neuropathic pain.

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling.

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5-S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.

Matrix metalloproteinase-14 both sheds cell surface neuronal glial antigen 2 (NG2) proteoglycan on macrophages and governs the response to peripheral nerve injury.

Neuronal glial antigen 2 (NG2) is an integral membrane chondroitin sulfate proteoglycan expressed by vascular pericytes, macrophages (NG2-Mφ), and progenitor glia of the nervous system. Herein, we revealed that NG2 shedding and axonal growth, either independently or jointly, depended on the pericellular remodeling events executed by membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14). Using purified NG2 ectodomain constructs, individual MMPs, and primary NG2-Mφ cultures, we demonstrated for the first time that MMP-14 performed as an efficient and unconventional NG2 sheddase and that NG2-Mφ infiltrated into the damaged peripheral nervous system. We then characterized the spatiotemporal relationships among MMP-14, MMP-2, and tissue inhibitor of metalloproteinases-2 in sciatic nerve. Tissue inhibitor of metalloproteinases-2-free MMP-14 was observed in the primary Schwann cell cultures using the inhibitory hydroxamate warhead-based MP-3653 fluorescent reporter. In teased nerve fibers, MMP-14 translocated postinjury toward the nodes of Ranvier and its substrates, laminin and NG2. Inhibition of MMP-14 activity using the selective, function-blocking DX2400 human monoclonal antibody increased the levels of regeneration-associated factors, including laminin, growth-associated protein 43, and cAMP-dependent transcription factor 3, thereby promoting sensory axon regeneration after nerve crush. Concomitantly, DX2400 therapy attenuated mechanical hypersensitivity associated with nerve crush in rats. Together, our findings describe a new model in which MMP-14 proteolysis regulates the extracellular milieu and presents a novel therapeutic target in the damaged peripheral nervous system and neuropathic pain.

The calcium-binding proteins S100A8 and S100A9 initiate the early inflammatory program in injured peripheral nerves.

To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve.

Spinal activity of interleukin 6 mediates myelin basic protein-induced allodynia.

Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting saltatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e., mechanical allodynia). Our earlier (Liu et al., 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84-104) into an intact sciatic nerve produces a robust and long-lasting (>30days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel α2δ1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system.

Activatable and Cell-Penetrable Multiplex FRET Nanosensor for Profiling MT1-MMP Activity in Single Cancer Cells.

We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.

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.

The alternatively spliced fibronectin CS1 isoform regulates IL-17A levels and mechanical allodynia after peripheral nerve injury.

BACKGROUND: Mechanical pain hypersensitivity associated with physical trauma to peripheral nerve depends on T-helper (Th) cells expressing the algesic cytokine, interleukin (IL)-17A. Fibronectin (FN) isoform alternatively spliced within the IIICS region encoding the 25-residue-long connecting segment 1 (CS1) regulates T cell recruitment to the sites of inflammation. Herein, we analyzed the role of CS1-containing FN (FN-CS1) in IL-17A expression and pain after peripheral nerve damage. METHODS: Mass spectrometry, immunoblotting, and FN-CS1-specific immunofluorescence analyses were employed to examine FN expression after chronic constriction injury (CCI) in rat sciatic nerves. The acute intra-sciatic nerve injection of the synthetic CS1 peptide (a competitive inhibitor of the FN-CS1/α4 integrin binding) was used to elucidate the functional significance of FN-CS1 in mechanical and thermal pain hypersensitivity and IL-17A expression (by quantitative Taqman RT-PCR) after CCI. The CS1 peptide effects were analyzed in cultured primary Schwann cells, the major source of FN-CS1 in CCI nerves. RESULTS: Following CCI, FN expression in sciatic nerve increased with the dominant FN-CS1 deposition in endothelial cells, Schwann cells, and macrophages. Acute CS1 therapy attenuated mechanical allodynia (pain from innocuous stimulation) but not thermal hyperalgesia and reduced the levels of IL-17A expression in the injured nerve. CS1 peptide inhibited the LPS- or starvation-stimulated activation of the stress ERK/MAPK pathway in cultured Schwann cells. CONCLUSIONS: After physical trauma to the peripheral nerve, FN-CS1 contributes to mechanical pain hypersensitivity by increasing the number of IL-17A-expressing (presumably, Th17) cells. CS1 peptide therapy can be developed for pharmacological control of neuropathic pain.

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.

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.