Platelet-derived growth factor and its role in health and disease

Platelet-derived growth factor (PDGF) was first discovered in platelets because they are the principal source of mitogenic activity in whole blood serum for mesenchymal cells in culture. PDGF is ubiquitous in that it can be formed by a large number of normal cells as well as many varieties of transformed cells. However, its expression and biological activity appear to be controlled at a number of different levels. The molecule consists of two peptide chains (termed 'A' and 'B') and is found as one of at least three possible isoforms, (AB, AA or BB). Each of these isoforms binds to a high-affinity cell-surface receptor that is composed of two different subunits, each of which has specificity for one or the other of the peptide chains of PDGF. The two receptor subunits are present in differing amounts on different cell types, and therefore the capacity of the different isoforms of PDGF to induce mitogenesis depends on the specific PDGF isoform and the relative numbers of receptor subunits present on the responding cell. In addition to inducing cell replication, PDGF elicits a number of intracellular signals related to mitogenesis, is chemotactic, is a vasoconstrictor, activates leukocytes, and modulates extracellular matrix turnover. This growth factor is probably involved in a number of biologically important events including wound repair, embryogenesis and development, and inflammation, leading to fibrosis, atherosclerosis and neoplasia.     

Contribution of active-site residues to the function of onconase, a ribonuclease with antitumoral activity

Onconase (ONC), a homologue of ribonuclease A (RNase A), is in clinical trials for the treatment of cancer. ONC possesses a conserved active-site catalytic triad, which is composed of His10, Lys31, and His97. The three-dimensional structure of ONC suggests that two additional residues, Lys9 and an N-terminal lactam formed from a glutamine residue (Pca1), could also contribute to catalysis. To determine the role of Pca1, Lys9, and Lys31 in the function of ONC, site-directed mutagenesis was used to replace each with alanine. Values of k(cat)/K(M) for the variants were determined with a novel fluorogenic substrate, which was designed to match the nucleobase specificity of ONC and gives the highest known k(cat)/K(M) value for the enzyme. The K9A and K31A variants display 10(3)-fold lower k(cat)/K(M) values than the wild-type enzyme, and a K9A/K31A double variant suffers a >10(4)-fold decrease in catalytic activity. In addition, replacing Lys9 or Lys31 eliminates the antitumoral activity of ONC. The side chains of Pca1 and Lys9 form a hydrogen bond in crystalline ONC. Replacing Pca1 with an alanine residue lowers the catalytic activity of ONC by 20-fold. Yet, replacing Pca1 in the K9A variant enzyme does not further reduce catalytic activity, revealing that the function of the N-terminal pyroglutamate residue is to secure Lys9. The thermodynamic cycle derived from k(cat)/K(M) values indicates that the Pca1...Lys9 hydrogen bond contributes 2.0 kcal/mol to the stabilization of the rate-limiting transition state during catalysis. Finally, binding isotherms with a substrate analogue indicate that Lys9 and Lys31 contribute little to substrate binding and that the low intrinsic catalytic activity of ONC originates largely from the low affinity of the enzyme for its substrate. These findings could assist the further development of ONC as a cancer chemotherapeutic.     

Physiological quiescence in plasma-derived serum: Influence of platelet-derived growth factor on cell growth in culture

A platelet-derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma-derived serum lacks such platelet-derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma-derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma-derived serum) and growing state (5% whole blood serum or 5% plasma-derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma-derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations: G₁ = 5.6 hours; S = 7.6 hours; and G₂ + M = 3.8 hours. Based on these studies the argument is presented that cells cultured in 5% plasma-derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma-derived serum represents an appropriate state to examine the perturbation of quiescent cells.     

Catalysis of imido group hydrolysis in a maleimide conjugate

Maleimides are often used for biomolecular conjugation with thiols. An underappreciated aspect of the imido group in a maleimide conjugate is its susceptibility to spontaneous hydrolysis, resulting in undesirable heterogeneity. Here, a chromophoric maleimide is used to demonstrate that both molybdate and chromate catalyze the hydrolysis of an imido group near neutral pH. Tungstate and 4-(dimethylamino)pyridine are less effective as catalysts. This work reveals a new mode of chemical reactivity for molybdate and chromate, and provides a strategy for decreasing the heterogeneity of bioconjugates derived from maleimides.     

Human Ribonuclease with a Pendant Poly(Ethylene Glycol) Inhibits Tumor Growth in Mice

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-CPEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 μmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ~5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.     

The role of charge in lipid selectivity for the nicotinic acetylcholine receptor.

The effect of salt and pH titration on the selectivity of spin-labeled analogues of phosphatidic acid, phosphatidylserine, phosphatidylcholine, and stearic acid for the nicotinic acetylcholine receptor (nAcChoR) reconstituted into dioleoylphosphatidylcholine was examined at 0 degrees C using electron spin resonance spectroscopy. The order of selectivity at pH 7.4 and 0 mM NaCl was phosphatidylserine > stearic acid > phosphatidic acid > phosphatidylcholine. The addition up to 2 M NaCl or titration of pH from 5.0 to > 9.0 did not alter the selectivity of the phospholipids for the nAcChoR. For stearic acid, conversely, titration of pH from 5.0 to 9.0 at 0 mM NaCl and titration of NaCl from 0 to 2 M at pH 9.0 both increased selectivity for the nAcChoR. It is concluded that electrostatic interactions do not account for the selectivity of the negatively charged phospholipids, phosphatidylserine, and phosphatidic acid for the nAcChoR. This is consistent with the known orientation of the transmembrane sequences M1 and M4, which predicts a balance in the number of negative and positive charges in the lipid-protein interface and suggests that the two positive charges on each M3 helix are not exposed to the lipid-protein interface.     

Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer

Bovine seminal ribonuclease (BS-RNase) is a homologue of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, BS-RNase has notable toxicity for human tumor cells. Wild-type BS-RNase is a homodimer linked by two intermolecular disulfide bonds. This quaternary structure endows BS-RNase with resistance to inhibition by the cytosolic ribonuclease inhibitor protein (RI), which binds tightly to RNase A and monomeric BS-RNase. Here, we report on the creation and analysis of monomeric variants of BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type BS-RNase, suggesting that the cytotoxicity of the wild-type enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of BS-RNase. Moreover, BS-RNase monomers described herein are more toxic to human tumor cells than is any known variant or homologue of RNase A including Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable malignant mesothelioma.     

Structure and stability of the P93G variant of ribonuclease A.

The peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 A. This structure is essentially identical to that of the wild-type protein, except for the 91-94 beta-turn containing the substitution. In the wild-type protein, the beta-turn is of type VIa. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of delta deltaGm which reports on the stability lost in the variants, is 1.5-fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.