PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B.

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their ß1 and ß3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-ß (PDGF Receptor ß) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-ß transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

Activation of the PDGF ß Receptor by a Persistent Artificial Signal Peptide.

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF ß receptor (PDGFßR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFßR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFßR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFßR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFßR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFßR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.

Structural insights into the modulation of PDGF/PDGFR-ß complexation by hyaluronan derivatives.

Angiogenesis is an important physiological process playing a crucial role in wound healing and cancer progression. Vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are key players in angiogenesis. Based on previous findings regarding the modulation of VEGF activity by glycosaminoglycans (GAG), here we explore the interaction of hyaluronan (HA)-based GAG with PDGF and its receptor PDGFR-ß by applying molecular modeling and dynamics simulations in combination with surface plasmon resonance (SPR). Computational analysis on the interaction of oligo-hyaluronan derivatives with different sulfation pattern and functionalization shows that these GAG interact with PDGF in relevant regions for receptor recognition, and that high sulfation as well as modification with the TAMRA group convey stronger binding. On the other hand, the studied oligo-hyaluronan derivatives are predicted to scarcely recognize PDGFR-ß. SPR results are in line with the computational predictions regarding the binding pattern of HA tetrasaccharide (HA4) derivatives to PDGF and PDGFR-ß. Furthermore, our experimental results also show that the complexation of PDGF to PDGFR-ß can be modulated by HA4 derivatives. The results found open the path for considering HA4 derivatives as potential candidates to be exploited for modulation of the PDGF/PDGFR-ß signaling system in angiogenesis and related disease conditions.

Platelet-derived growth factor receptor beta activates Abl2 via direct binding and phosphorylation.

Abl family kinases are nonreceptor tyrosine kinases activated by diverse cellular stimuli that regulate cytoskeleton organization, morphogenesis, and adhesion. The catalytic activity of Abl family kinases is tightly regulated in cells by a complex set of intramolecular and intermolecular interactions and post-translational modifications. For example, the platelet-derived growth factor receptor beta (PDGFRß), important for cell proliferation and chemotaxis, is a potent activator of Abl family kinases. However, the molecular mechanism by which PDGFRß engages and activates Abl family kinases is not known. We show here that the Abl2 Src homology 2 domain directly binds to phosphotyrosine Y771 in the PDGFRß cytoplasmic domain. PDGFRß directly phosphorylates multiple novel sites on the N-terminal half of Abl2, including Y116, Y139, and Y161 within the Src homology 3 domain, and Y299, Y303, and Y310 on the kinase domain. Y116, Y161, Y272, and Y310 are all located at or near the Src homology 3/Src homology 2-kinase linker interface, which helps maintain Abl family kinases in an autoinhibited conformation. We also found that PDGFRß-mediated phosphorylation of Abl2 in vitro activates Abl2 kinase activity, but mutation of these four tyrosines (Y116, Y161, Y272, and Y310) to phenylalanine abrogated PDGFRß-mediated activation of Abl2. These findings reveal how PDGFRß engages and phosphorylates Abl2 leading to activation of the kinase, providing a framework to understand how growth factor receptors engage and activate Abl family kinases.

The Master of Puppets: Pleiotropy of PDGFRB and its Relationship to Multiple Diseases.

The platelet-derived growth factor receptor beta (PDGFRB) gene is involved in proliferative and developmental processes in mammals. Variations in this gene lead to several different syndromic conditions, such as infantile myofibromatosis I, sporadic port-wine stain, primary familial brain calcification, and the Penttinen and overgrowth syndromes. Our objective was to investigate PDGFRB's genetic relationship to clinical conditions and evaluate the protein interactions using GeneNetwork, GeneMANIA, and STRING network databases. We have evidenced the gene's pleiotropy through its many connections and its link to syndromic conditions. Therefore, PDGFRB may be an important therapeutic target for treating such conditions.

Biologically Active Ultra-Simple Proteins Reveal Principles of Transmembrane Domain Interactions.

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor ß receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions.

Dynamin inhibitors impair platelet-derived growth factor ß-receptor dimerization and signaling.

The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor ß-receptor (PDGFR-ß) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-ß preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-ß by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-ß activation, and that this link is not shared with the epidermal growth factor receptor.

Modulation of pericytes by a fusion protein comprising of a PDGFRß-antagonistic affibody and TNFα induces tumor vessel normalization and improves chemotherapy.

The delivery of anticancer drugs is hampered by tumor vessels with abnormal structure and function, which requires that vessel normalization be mediated by pharmaceutics. The current strategies for vessel normalization focus on direct modulation of endothelial cells (ECs), which frequently affect vessels in normal tissues. Modulating EC-supporting cells, such as pericytes (PCs), is a new direction. Here, we produced a fusion protein, Z-TNFα, by fusing the platelet-derived growth factor receptor ß (PDGFRß)- antagonistic affibody ZPDGFRß to tumor necrosis factor α (TNFα). Owing to the affinity of fused ZPDGFRß for PDGFRß, Z-TNFα binds PDGFRß+ PCs but not PDGFRß- ECs. Low-dose (1 µg/mouse) Z-TNFα treatment remodeled the tumor vessels, thus reducing vessel permeability and increasing vessel perfusion. As a result, the Z-TNFα treatment improved the delivery of doxorubicin (DOX) and enhanced its antitumor effect, indicating that Z-TNFα induced normalization of tumor vessels. Mechanically, the tumor vessel normalization mediated by Z-TNFα might be attributed to the reduction of vascular endothelial growth factor (VEGF) secretion by PCs and the elevated expression of intercellular cell adhesion molecule-1 (ICAM-1) in PCs, which might suppress the proliferation and migration of ECs and simultaneously trigger interaction between perivascular macrophages and PCs. These results demonstrated that tumor-associated PCs could be considered novel target cells for vessel normalization, and Z-TNFα might be developed as a potential tool for antitumor combination therapy.

Platelets activated by the anti-ß2GPI/ß2GPI complex release microRNAs to inhibit migration and tube formation of human umbilical vein endothelial cells.

BACKGROUND: Patients with anti-ß2GPI antibodies display significantly higher platelet activation/aggregation and vascular endothelial cell damage. The mechanism underlying the correlation between platelet activation, vascular endothelial cell dysfunctions and anti-ß2GPI antibodies remains unknown. METHODS: In this study, we derived miR-96 and -26a from platelets activated by the anti-ß2GPI/ß2GPI complex and explored their role in modulating human umbilical vein endothelial cell (HUVEC) migration and tube formation. RESULTS: Anti-ß2GPI/ß2GPI complex induces the release of platelet-derived microparticles (p-MPs). The amounts of miR-96 and -26a in these p-MPs were also higher than for the control group. Co-incubation of HUVECs with p-MPs resulted in the transfer of miR-96 and -26a into HUVECs, where they inhibited migration and tube formation. The targeting role of these miRNAs was further validated by directly downregulating targeted selectin-P (SELP) and platelet-derived growth factor receptor alpha (PDGFRA) via luciferase activity assay. CONCLUSION: Our study suggests that miR-96 and -26a in p-MPs can inhibit HUVEC behavior by targeting SELP and PDGFRA.

Effect of PDGF-B aptamer on PDGFRß/PDGF-B interaction: Molecular dynamics study.

PDGFRß/PDGF-B interaction plays a role in angiogenesis, and is mandatory in wound healing and cancer treatment. It has been reported that the PDGF-B aptamer was able to bind to PDGF-B, thus regulating the angiogenesis. However, the binding interaction between the aptamer and the growth factor, including the binding sites, has not been well investigated. This study applied a molecular dynamics (MD) simulation to investigate the aptamer-growth factor interaction in the presence or absence of a receptor (PDGFRß). Characterization of the structure of an aptamer-growth factor complex revealed binding sites from each section in the complex. Upon the complex formation, PDGF-B and its aptamer exhibited less flexibility in their molecular movement, as indicated by the minimum values of RMSD, RMSF, loop-to-loop distance, and the summation of PCA eigenvalues. Our study of residue pairwise interaction demonstrated that the binding interaction was mainly contributed by electrostatic interaction between the positively-charged amino acid and the negatively-charged phosphate backbone. The role of the PDGF-B aptamer in PDGFRß/PDGF-B interaction was also investigated. We demonstrated that the stability of the Apt-PDGF-B complex could prevent the presence of a competitor, of PDGFRß, interrupting the binding process. Because the aptamer was capable of binding with PDGF-B, and blocking the growth factor from the PDGFRß, it could down regulate the consequent signaling pathway. We provide evidence that the PDGF-BB aptamer is a promising molecule for regulation of angiogenesis. The MD study provides a molecular understanding to modification of the aptamer binding interaction, which could be used in a number of medical applications.

The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor ß Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression.

The platelet-derived growth factor receptor ß (PDGFR-ß) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-ß promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-ß NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-ß pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-ß is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-ß NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-ß NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-ß promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-ß i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-ß promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-ß-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.

Optimization of the tumor microenvironment and nanomedicine properties simultaneously to improve tumor therapy.

Effective delivery of nanomedicines to tumor tissues depends on both the tumor microenvironment and nanomedicine properties. Accordingly, tumor microenvironment modification or advanced design of nanomedicine was emerging to improve nanomedicine delivery to tumors. However, few studies have emphasized the necessity to optimize the tumor microenvironment and nanomedicine properties simultaneously to improve tumor treatment. In the present study, imatinib mesylate (IMA) was used to normalize the tumor microenvironment including platelet-derived growth factor receptor-ß expression inhibition, tumor vessel normalization, and tumor perfusion improvement as demonstrated by immunofluorescence staining. In addition, the effect of tumor microenvironment normalization on tumor delivery of nanomedicines with different sizes was carefully investigated. It was shown that IMA treatment significantly reduced the accumulation of nanoparticles (NPs) around 110 nm but enhanced the accumulation of micelles around 23 nm by in vivo fluorescence imaging experiment. Furthermore, IMA treatment limited the distribution of NPs inside tumors but increased that of micelles with a more homogeneous pattern. Finally, the anti-tumor efficacy study displayed that IMA pretreatment could significantly increase the therapeutic effects of paclitaxel-loaded micelles. All-together, a new strategy to improve nanomedicine delivery to tumor was provided by optimizing both nanomedicine size and the tumor microenvironment simultaneously, and it will have great potential in clinics for tumor treatment.

Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension.

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.

Next-Generation Sequencing and In Silico Analysis Facilitate Prolonged Response to Pazopanib in a Patient With Metastatic Urothelial Carcinoma of the Renal Pelvis.

With the advent of widespread tumor genetic profiling, an increased number of mutations with unknown significance are being identified. Often, a glut of uninterpretable findings may confuse the clinician and provide little or inappropriate guidance in therapeutic decision-making. This report describes a method of protein modeling by in silico analysis (ie, using computer simulation) that is easily accessible to the practicing clinician without need for further laboratory analysis, which can potentially serve as a guide in therapeutic decisions based on poorly characterized tumor mutations. An example of this model is given wherein poorly characterized KIT, PDGFRB, and ERBB2 mutations were discovered in a patient with treatment-refractory metastatic transitional cell carcinoma of the renal pelvis. The KIT and PDGFRB mutations were predicted to be pathogenic using in silico analysis, whereas the ERBB2 mutation was predicted to be benign. Based on these findings, the patient was treated with pazopanib and achieved a partial response that lasted for 7.5 months. We propose that in silico analysis be explored as a potential means to further characterize genetic abnormalities found by tumor profiling assays, such as next-generation sequencing.

Structure of Full-Length Human PDGFRß Bound to Its Activating Ligand PDGF-B as Determined by Negative-Stain Electron Microscopy.

Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the unregulated states, cancer formation. One long-standing puzzle regarding RTKs centers on how the extracellular domain (ECD), which detects and binds to growth factors, is coupled with the intracellular domain kinase activation. While extensive structural works on the soluble portions of RTKs have provided critical insights into RTK structures and functions, lack of a full-length receptor structure has hindered a comprehensive overview of RTK activation. In this study, we successfully purified and determined a 27-Å-resolution structure of PDGFRß [a full-length human platelet-derived growth factor receptor], in complex with its ligand PDGF-B. In the ligand-stimulated complex, two PDGFRßs assemble into a dimer via an extensive interface essentially running along the full-length of the receptor, suggesting that the membrane-proximal region, the transmembrane helix and the kinase domain of PDGFRß are involved in dimerization. Major structural differences are seen between the full-length and soluble ECD structures, rationalizing previous experimental data on how membrane-proximal domains modulate receptor ligand-binding affinity and dimerization efficiency. Also, in contrast to the 2-fold symmetry of the ECD, the intracellular kinase domains adopt an asymmetric dimer arrangement, in agreement with prior observations for the closely related KIT receptor. In essence, the structure provides a first glimpse into how platelet-derived growth factor receptor ECD, upon ligand stimulation, is coupled to its intracellular domain kinase activation.

Design, Synthesis, and Characterization of a Photoactivatable Caged Prodrug of Imatinib.

Imatinib is the first protein kinase inhibitor approved for clinical use and is a seminal drug for the concept of targeted therapy. Herein we report on the design, synthesis, photokinetic properties, and in vitro enzymatic evaluation of a photoactivatable caged prodrug of imatinib. This approach allows spatial and temporal control over the activation of imatinib triggered by ultraviolet light. The successful application of the photoactivation concept to this significant kinase inhibitor provides further evidence for the caging technique as a feasible approach in the kinase field. The presented photoactivatable imatinib prodrug will be highly useful as a pharmacological tool to study the impact of imatinib toward biological systems in greater detail.

Mechanisms of PDGFRalpha promiscuity and PDGFRbeta specificity in association with PDGFB.

Platelet-derived growth factor receptor alpha (PDGFRalpha) interacts with PDGFs A, B, C and AB, while PDGFRbeta binds to PDGFs B and D, thus suggesting that PDGFRalpha is more promiscuous than PDGFRbeta. The structural analysis of PDGFRalpha-PDGFA and PDGFRalpha-PDGFB complexes and a molecular explanation for the promiscuity of PDGFRalpha and the specificity of PDGFRbeta remain unclear. In the present study, we modeled the three extracellular domains of PDGFRalpha using a previous crystallographic structure of PDGFRbeta as a template. Additionally, we analyzed the interacting residues of PDGFRalpha-PDGFA and PDGFRalpha-PDGFB complexes using docking simulations. The validation of the resulting complexes was evaluated by molecular dynamics simulations. Structural analysis revealed that changes of non-aromatic amino acids in PDGFRalpha to aromatic amino acids in PDGFRbeta (I139F, P267F and N204Y) may be involved in the promiscuity of PDGFRalpha. Indeed, substitution of amino acids with few probabilities of rotamer changes in PDGFRbeta (M133A, N163E and N179S) and energy stability due to the formation of hydrogen bond in PDGFRbeta could explain the specificity of PDGFRbeta. These results may be used as an input for a better and more specific drug and peptide design targeting diseases related with the malfunction of PDGFs and PDGFRalpha such as cancer and atherosclerosis.