Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface

Involvement of chondroitin sulfate E in the liver tumor focal formation of murine osteosarcoma cells

Cell surface heparan sulfate plays a critical role in regulating the metastatic behavior of tumor cells, whereas the role of chondroitin sulfate/dermatan sulfate (CS/DS) has been little understood in this context. Here, we characterized CS/DS chains from the murine osteosarcoma cell line LM8G7, which forms tumor nodules in liver. Structural analysis of the CS/DS chains showed a higher proportion of GlcUAβ1-3GalNAc(4,6-O-disulfate) (E-units) in LM8G7 (12%) than in its parental cell line LM8 (6%), which rarely forms tumors in the liver. Immunostaining with GD3G7, an antibody specific to E-units, confirmed the higher expression of the epitope in LM8G7 than LM8 cells. The tumor focal formation of LM8G7 cells in the liver in mice was effectively inhibited by the pre-administration of CS-E (rich in E-unit) or the pre-incubation of the antibody GD3G7 with the tumor cells. CS-E or GD3G7 inhibited the adhesion of LM8G7 cells to a laminin-coated plate in vitro. In addition, the invasive ability of LM8G7 cells in vitro was also reduced by the addition of CS-E or the antibody. Further, CS-E or the antibody inhibited the proliferation of LM8G7 cells dose-dependently. The binding of LM8G7 cells to VEGF in vitro was also significantly reduced by CS-E and GD3G7. Thus, the present study reveals the significance of highly sulfated CS/DS structures in the liver colonization of osteosarcoma cells and also provides a framework for the development of GAG-based anti-cancer molecules

Tumor penetrating peptides

Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the blood

Sequence dependence of C-end rule peptides in binding and activation of > neuropilin-1 receptor

Neuropilin-1 (NRP-1) is a hub receptor that plays an essential role in angiogenesis and vascular permeability. It is over-expressed in the new blood vessels grown by tumor cells and is a target for anti-tumor treatments. Peptides that expose the consensus sequence R/K/XXR/K at the C-terminus (C-end rule or CendR peptides) bind to NRP-1 and are internalized into the cell. We used peptide phage display binding assays and molecular dynamics (MD) simulations to study the potential role of the central residues of CendR peptides in binding and activation of the NRP-1 receptor. The high stability of RPAR–receptor domain complex stems from the formation of a characteristic pattern of three hydrogen bonds between the peptide C-terminus and the residues in the NRP-1 loop III. Any changes in the peptide structure that fail to preserve this triad result in a less-stable complex. We performed a systematic study of RXXR mutants, where X = A/D/S/R/P, in order to test the effect of replacement of A or P on the binding capabilities. Our results, both experimental and computational, show that RRAR, RDAR, RPDR, RPRR and RPPR are capable of binding NRP-1. However, only RPPR and RPRR segments form an optimal organization around loop III with low potential energy. In other analogs, the absence of these stabilizing interactions always results in higher potential energy of the complexes. The binding of RPAR analogs does not guarantee receptor activation; only stable complexes that are properly stabilized via loop III appear able to trigger NRP-1 activation.Postprint (published version

Journal of Structural Biology

Neuropilin-1 (NRP-1) is a hub receptor that plays an essential role in angiogenesis and vascular perme- ability. It is over-expres sed in the new blood vessels grown by tumor cells and is a target for anti-tumor treatment s. Peptides that expose the consensus sequence R/K/ XX R/K at the C-terminus ( C-end rule or CendR peptides) bind to NRP-1 and are internalized into the cell. We used peptide phage display binding assays and molecular dynamics (MD) simulations to study the potential role of the central residues of CendR peptides in binding and activa tion of the NRP-1 receptor. The high stability of RPAR–receptor domain complex stems from the formation of a characteristic pattern of three hydrogen bonds between the peptide C-terminus and the residues in the NRP-1 loop III. Any changes in the peptide structure that fail to preserve this triad result in a less-stable complex. We performed a systematic study of R XX R mutants, where X = A/D/S/R/P, in order to test the effect of replacement of A or P on the binding capabil- ities. Our results, both experimental and computational, show that RRAR, RDAR, RPDR, RPRR and RPPR are capable of binding NRP-1. However, only RPPR and RPRR segments form an optimal organization around loop III with low potential energy. In other analogs, the absence of these stabilizing interactions always results in higher potential energy of the complexes. The binding of RPAR analogs does not guarantee receptor activation; only stable complexes that are properly stabilized via loop III appear able to trigger NRP-1 activati onPeer ReviewedPostprint (published version

Journal of Structural Biology

Neuropilin-1 (NRP-1) is a hub receptor that plays an essential role in angiogenesis and vascular perme- ability. It is over-expres sed in the new blood vessels grown by tumor cells and is a target for anti-tumor treatment s. Peptides that expose the consensus sequence R/K/ XX R/K at the C-terminus ( C-end rule or CendR peptides) bind to NRP-1 and are internalized into the cell. We used peptide phage display binding assays and molecular dynamics (MD) simulations to study the potential role of the central residues of CendR peptides in binding and activa tion of the NRP-1 receptor. The high stability of RPAR–receptor domain complex stems from the formation of a characteristic pattern of three hydrogen bonds between the peptide C-terminus and the residues in the NRP-1 loop III. Any changes in the peptide structure that fail to preserve this triad result in a less-stable complex. We performed a systematic study of R XX R mutants, where X = A/D/S/R/P, in order to test the effect of replacement of A or P on the binding capabil- ities. Our results, both experimental and computational, show that RRAR, RDAR, RPDR, RPRR and RPPR are capable of binding NRP-1. However, only RPPR and RPRR segments form an optimal organization around loop III with low potential energy. In other analogs, the absence of these stabilizing interactions always results in higher potential energy of the complexes. The binding of RPAR analogs does not guarantee receptor activation; only stable complexes that are properly stabilized via loop III appear able to trigger NRP-1 activati onPeer Reviewe

Journal of Structural Biology

Neuropilin-1 (NRP-1) is a hub receptor that plays an essential role in angiogenesis and vascular perme- ability. It is over-expres sed in the new blood vessels grown by tumor cells and is a target for anti-tumor treatment s. Peptides that expose the consensus sequence R/K/ XX R/K at the C-terminus ( C-end rule or CendR peptides) bind to NRP-1 and are internalized into the cell. We used peptide phage display binding assays and molecular dynamics (MD) simulations to study the potential role of the central residues of CendR peptides in binding and activa tion of the NRP-1 receptor. The high stability of RPAR–receptor domain complex stems from the formation of a characteristic pattern of three hydrogen bonds between the peptide C-terminus and the residues in the NRP-1 loop III. Any changes in the peptide structure that fail to preserve this triad result in a less-stable complex. We performed a systematic study of R XX R mutants, where X = A/D/S/R/P, in order to test the effect of replacement of A or P on the binding capabil- ities. Our results, both experimental and computational, show that RRAR, RDAR, RPDR, RPRR and RPPR are capable of binding NRP-1. However, only RPPR and RPRR segments form an optimal organization around loop III with low potential energy. In other analogs, the absence of these stabilizing interactions always results in higher potential energy of the complexes. The binding of RPAR analogs does not guarantee receptor activation; only stable complexes that are properly stabilized via loop III appear able to trigger NRP-1 activati onPeer Reviewe