Kenneth E. Neet

[8] Hysteretic enzymes

Publisher Summary This chapter discusses that the concept that enzymic reactions demonstrate linear reaction rates and, therefore, linear progress curves, after a very rapid attainment of the steady state, dates to the early formulations of enzyme kinetics. It also discusses the analysis of the steady-state kinetics in such a system, the possibilities of producing cooperative kinetics in a monomeric enzyme, the possibility of altering the kinetic cooperativity of an oligomeric enzyme, and the correlation between enzymes showing hysteretic responses and possessing cooperativity in their kinetics. The chapter presents a unified picture of hysteretic enzymes, the areas of agreement between different workers, and the details of certain specific models that make them attractive to explain particular enzymes. Two aspects of these systems discussed are the hysteretic (slow) process itself and its relationship to cooperativity in the steady-state kinetics. Although these two aspects are related through the models, they must be remembered as different concepts; hysteresis may occur without cooperativity, but cooperativity (by this mechanism) requires a molecular transition.

Nerve growth factor: structure, function and therapeutic implications for Alzheimer's disease.

Over the past decade, neurotrophic factors have generated much excitement for their potential as therapy for neurological disorders. In this regard, nerve growth factor (NGF), the founding member of the neurotrophin family, has generated great interest as a potential target for the treatment of Alzheimers disease (AD). This interest is based on the observation that cholinergic basal forebrain (CBF) neurons which provide the major source of cholinergic innervation to the cerebral cortex and hippocampus undergo selective and severe degeneration in advanced AD and that these neurons are dependent upon NGF and its receptors for their survival. In fact, NGF transduces its effects by binding two classes of cell surface receptors, TrkA and p75(NTR), both of which are produced by CBF neurons. This review focuses on NGF/receptor binding, signal transduction, regulation of specific cellular endpoints, and the potential use of NGF in AD. Alterations in NGF ligand and receptor expression at different stages of AD are summarized. Recent results suggest that cognitive deficits in early AD and mild cognitive impairment (MCI) are not associated with a cholinergic deficit. Thus, the earliest cognitive deficits in AD may involve brain changes other than simply cholinergic system dysfunction. Recent findings indicate an early defect in NGF receptor expression in CBF neurons; therefore treatments aimed at facilitating NGF actions may prove highly beneficial in counteracting the cholinergic dysfunction found in end-stage AD and attenuating the rate of degeneration of these cholinergic neurons.

[20] Cooperativity in enzyme function: Equilibrium and kinetic aspects

Publisher Summary Cooperativity in protein or enzyme systems commonly refers to ligand binding, with the type of positive cooperativity, that gives rise to the sigmoid curves of oxygen binding to hemoglobin. However, the concept and the practical applications are much more encompassing than that single interpretation. Cooperativity in its broadest sense includes protein folding-unfolding reactions (e.g., denaturation, helix-coil transitions), macromolecular assembly (e.g., tubulin, sickling of hemoglobin S), binding of proteins as ligands to DNA (e.g., transcriptional factors, single-stranded DNA binding proteins), and binding of proteins as ligands to the membrane receptors (e.g., hormones, growth factors). Cooperativity, in general, is any process in which the initial event (hydrogen bonding, protein-protein interaction, ligand binding) affects subsequent similar events, in the cases cited via communication through intra- or intermolecular protein interactions. Protein structures are uniquely suited for the ability to communicate the information needed for cooperativity through conformational changes and intersubunit binding affinities. This chapter not only discusses the substrate and ligand binding to enzymes, but also briefly discusses the extension to proteins as ligands binding to other macromolecular systems. The current discussion of cooperativity describes the basics of enzyme cooperativity, emphasizes the advances since the early 1980s, and utilizes the examples of several systems to describe how cooperativity is currently studied and analyzed.

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75NTR. During retinal injury, endogenous NGF, TrkA, and p75NTR are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75NTR affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75NTR agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75NTR action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α2-macroglobulin. Antagonists of p75NTR inhibit TNF-α and α2-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

Gene 5 protein of bacteriophage fd: A dimer which interacts Co-operatively with DNA☆

Abstract Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein. Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated. The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)4], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)8], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented.

Proneurotrophins Require Endocytosis and Intracellular Proteolysis to Induce TrkA Activation

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.

Studies of the self-association of bacteriophage T4 gene 32 protein by equilibrium sedimentation.

Abstract The self-association of the bacteriophage T4 gene 32 protein has been examined in the analytical ultracentrifuge under varying conditions to determine the nature of the process. The process is not a simple indefinite association with one association constant (monomer dimer trimer etc.). The complexity of the process is shown by (1) peculiarities in the molecular weight versus concentration curves, in the region of the dimer (observed with increasing ionic strength, at pH 10, in 0.04 m -MgCl2, with aged preparations, at 19 °C and in the presence of the oligonucleotide d(pT)10), (2) the increased sigmoidicity of the association curve in the presence of glycerol or oligo[d(pT)4], and (3) the discontinuity in the association curve at the tetramer at a pH value of approximately 9.4. A model with two association constants which could vary independently (monomer dimer tetramer etc.) explained many of the findings. However, a more complex model was required to explain curves which had a plateau at the dimer with increased association at higher protein concentrations. Thus, under all conditions examined there is evidence for more than one type of protein-protein interaction. These different interactions may be involved in a physiological function such as recombination.

Distinction between differentiation, cell cycle, and apoptosis signals in PC12 cells by the nerve growth factor mutant Δ9/13, which is selective for the p75 neurotrophin receptor

The common neurotrophin receptor p75NTR (low affinity nerve growth factor receptor) participates in the high‐affinity binding with the TrkA nerve growth factor (NGF) receptor, may mediate apoptosis, and may signal independently in a cell‐specific manner. The potential of p75NTR to signal independently of TrkA was investigated with an NGF mutant protein (NGFΔ9/13) that binds poorly to TrkA (Woo et al. [1995] J. Biol. Chem. 270:6278–6285). The NGFΔ9/13 mutant does not activate TrkA autophosphorylation and fails to stimulate the normal NGF‐induced growth arrest, demonstrating that TrkA activation is required to arrest PC12 cells at the NGF‐activated G1/S cell cycle checkpoint. However, apoptosis is successfully blocked and cell survival is promoted by the NGFΔ9/13 mutant in naive PC12 cells after serum withdrawal, suggesting that p75NTR can signal for survival autonomously of TrkA. Annexin V binding, an indication of apoptotic plasma membrane disruption, is inhibited by both NGF and the NGFΔ9/13 mutant after serum deprivation. Both NGF and the NGFΔ9/13 mutant inhibit the rapid apoptotic internucleosomal DNA cleavage of PC12 cells upon serum deprivation. Furthermore, the level of caspase3‐like activity that is rapidly activated by serum withdrawal from PC12 cells is reduced by both the NGFΔ9/13 protein and NGF. Finally, upon serum withdrawal, both NGF and the NGFΔ9/13 mutant activate nuclear translocation of the transcriptional factor NF‐κB (nuclear factor κB), a process involved in cell survival. These results are consistent with p75NTR inhibition of caspase‐mediated apoptosis in PC12 cells. The different physiologic responses elicited by NGFΔ9/13 indicate the potential for individual signaling by the two NGF receptors and also demonstrate the utility of NGF mutants for receptor‐selective signal transduction. J. Neurosci. Res. 63:10–19, 2001.

Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization

Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75NTR) and sortilin. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus). Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. Here we describe the construction, expression, and purification of this more stable proNGF molecule. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Expression was performed in stably transfected Sf21 insect cells. Purification involved strong cation-exchange chromatography and N60 immunoaffinity column chromatography. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells.