Effect of Zymogen Domains and Active Site Occupation on Activation of Prothrombin by von Willebrand Factor-binding Protein

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH₂-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1–263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1–474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1–474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1–263) or vWbp(1–474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1–263) and vWbp(1–474), with a 30–45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with d-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K_D values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.     

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.     

Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis

Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin⁺ intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ⁺ cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-β1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis.     

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

Tissue inhibitor of metalloproteinases-3 (TIMP-3) plays a key role in regulating extracellular matrix turnover by inhibiting matrix metalloproteinases (MMPs), adamalysins (ADAMs), and adamalysins with thrombospondin motifs (ADAMTSs). We demonstrate that levels of this physiologically important inhibitor can be regulated post-translationally by endocytosis. TIMP-3 was endocytosed and degraded by a number of cell types including chondrocytes, fibroblasts, and monocytes, and we found that the endocytic receptor low density lipoprotein receptor-related protein-1 (LRP-1) plays a major role in TIMP-3 internalization. However, the cellular uptake of TIMP-3 significantly slowed down after 10 h due to shedding of LRP-1 from the cell surface and formation of soluble LRP-1 (sLRP-1)-TIMP-3 complexes. Addition of TIMP-3 to HTB94 human chondrosarcoma cells increased the release of sLRP-1 fragments of 500, 215, 160, and 110 kDa into the medium in a concentration-dependent manner, and all of these fragments were able to bind to TIMP-3. TIMP-3 bound to sLRP-1, which was resistant to endocytosis, retained its inhibitory activity against metalloproteinases. Extracellular levels of sLRP-1 can thus increase the half-life of TIMP-3 in the extracellular space, controlling the bioavailability of TIMP-3 to inhibit metalloproteinases.     

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X₃X₂X₁-aminomethylcoumarin) and by determining specificity constants (k_cat/K_m), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X₁ position, basic residues or Trp in the X₂ position, and Pro ≫ Ala > Trp in the X₃ position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.     

The Efficiency of Dentin Sialoprotein-Phosphophoryn Processing Is Affected by Mutations Both Flanking and Distant from the Cleavage Site

Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G⁴⁴⁷↓D⁴⁴⁸ cleavage site in DSP-PP₂₄₀ had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P₄ to P₄′ blocked, impaired, or enhanced DSP-PP₂₄₀ cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP₂₄₀ had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP₂₄₀ significantly modified the amount of PP₂₄₀ product generated from DSP-PP₂₄₀ precursor protein cleavage suggests that such mutation may affect the mineralization process.