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Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) 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 p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-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.

Afflicted neurons in Alzheimer disease have been shown to display an imbalance in the expression of TrkA and p75(NTR) at the cell surface, and administration of nerve growth factor (NGF) has been considered and attempted for treatment. However, wild-type NGF causes extensive elaboration of neurites while providing survival support. This study was aimed at developing recombinant NGF muteins that did not support neuritogenesis while maintaining the survival response. Critical residues were identified at the ligand-receptor interface by point mutagenesis that played a greater importance in neuritogenesis versus survival. By combining point mutations, two survival-selective recombinant NGF muteins, i.e./7-84-103 and KKE/7-84-103, were generated. Both muteins reduced neuritogenesis in PC12 (TrkA(+)/p75(NTR+)) cells by90%, while concurrently retaining near wild-type survival activity in MG139 (TrkA(+) only) and PCNA fibroblast (p75(NTR+)-only) cells. Additionally, survival in both naive and terminally differentiated PC12 cells was shown to be intermediate between NGF and negative controls. Dose-response curves with 7-84-103 showed that the differentiation curve was shifted by about 100-fold, whereas the EC(50) for survival was only increased by 3.3-fold. Surface plasmon resonance analysis revealed a 200-fold decrease in binding of 7-84-103 to TrkA. The retention of cell survival was attributed to maintenance of signaling through the Akt survival pathway with reduced MAPK signaling for differentiation. The effect of key mutations along the NGF receptor interface are transmitted inside the cell to enable the generation of survival-selective recombinant NGF muteins that may represent novel pharmacologic lead agents for the amelioration of Alzheimer disease.

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.

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.

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.

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 (p75 NTR ) 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.

The tumor suppressor protein p53 is a transcription factor that regulates the response to cellular insults such as DNA damage and growth factor withdrawal. Transcriptional activity of p53 requires post-translational modification by phosphorylation and acetylation. This study used site-specific antibodies to demonstrate that nerve growth factor (NGF) treatment of PC12 cells results in p53 deacetylation at lysine (Lys) 382. Histone deacetylase (HDAC) activity, measured by a direct fluorescent assay, was increased after NGF treatment and peaked before p53 deacetylation. Inhibition of HDAC by trichostatin blocked the deacetylation of p53 and its transcriptional activity toward a reporter gene construct. Comparison of PC12 with PC12 cells containing a temperature-sensitive, dominant-negative construct showed that p53 deacetylation required functional p53. Inhibitors of MAP kinase that block p53 transactivation and inhibitors of TrkA receptor also abolished HDAC activation, indicating that deacetylation of p53 is an NGF-dependent post-translational mechanism of p53 activation. Finally, NGF or serum withdrawal did not lead to p53 deacetylation. A model is proposed in which the acetylation status of Lys 382 of p53 discriminates between cell cycle arrest and apoptosis.

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 Alzheimer's 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.

Neurotrophins interact with two distinct classes of cell-surface receptors, the Trk receptor tyrosine kinase family and the common neurotrophin receptor p75(NTR). For many years, the biological role of p75(NTR) remained obscure, being relegated to modulating Trk binding of neurotrophins. Recently, the importance of p75(NTR) as a signaling receptor in itself has become increasingly clear. The signals initiated by p75(NTR) are likely to be as complex as those for the Trk family and probably depend on the cell system in which such signaling is being studied. In this study, all members of the neurotrophin family were demonstrated to be capable of stimulating p75(NTR)-mediated activation of the mitogen-activated protein kinase (MAPK) family (ERK1,2). This activation is rapid and transient, peaking at 5-15 min, depending on the cell system. The classical MAPK cascade consists of the reaction series Ras-Raf-MEK-MAPK. The p75(NTR)-induced MAPK activation is MEK dependent but Raf independent. This result implies that neurotrophin activation of p75(NTR) results in some cascade (as yet unknown) that bypasses Raf and converges on MEK to result in activation of MAPK. This activated MAPK is then able to translocate to the nucleus. The effect of this MAPK activation on cell survival is dependent on cell type. These results support the concept that signaling from the p75(NTR) receptor is more diverse and extensive than previously believed.

Proteomics focuses on the high throughput study of the expression, structure, interactions, and, to some extent, function of large numbers of proteins. A true understanding of the functioning of a living cell also requires a quantitative description of the stoichiometry, kinetics, and energetics of each protein complex in a cellular pathway. Classical molecular biophysical studies contribute to understanding of these detailed properties of proteins on a smaller scale than does proteomics in that individual proteins are usually studied. This perspective article deals with the role of biophysical methods in the study of proteins in the proteomic era. Several important physical biochemical methods are discussed briefly and critiqued from the standpoint of information content and data acquisition. The focus is on conformational changes and macromolecular assembly, the utility of dynamic and static structural data, and the necessity to combine experimental approaches to obtain a full functional description. The conclusions are that biophysical information on proteins is a useful adjunct to "standard" proteomic methods, that data can be obtained by high throughput technology in some instances, but that hypothesis-driven experimentation may frequently be required.

It has recently been shown that transition metal cations Zn2+ and Cu2+ bind to histidine residues of nerve growth factor (NGF) and other neurotrophins (a family of proteins important for neuronal survival) leading to their inactivation. Experimental data and theoretical considerations indicate that transition metal cations may destabilize the ionic form of histidine residues within proteins, thereby decreasing their pK(a) values. Because the release of transition metal cations and acidification of the local environment represent important events associated with brain injury, the ability of Zn2+ and Cu2+ to bind to neurotrophins in acidic conditions may alter neuronal death following stroke or as a result of traumatic injury. To test the hypothesis that metal ion binding to neurotrophins is influenced by pH, the effects of Zn2+ and Cu2+ on NGF conformation, receptor binding and NGF tyrosine kinase (trkA) receptor signal transduction were examined under conditions mimicking cerebral acidosis (pH range 5.5-7.4). The inhibitory effect of Zn2+ on biological activities of NGF is lost under acidic conditions. Conversely, the binding of Cu2+ to NGF is relatively independent of pH changes within the studied range. These data demonstrate that Cu2+ has greater binding affinity to NGF than Zn2+ at reduced pH, consistent with the higher affinity of Cu2+ for histidine residues. These findings suggest that cerebral acidosis associated with stroke or traumatic brain injury could neutralize the Zn2+-mediated inactivation of NGF, whereas corresponding pH changes would have little or no influence on the inhibitory effects of Cu2+. The importance of His84 of NGF for transition metal cation binding is demonstrated, confirming the involvement of this residue in metal ion coordination.

The common neurotrophin receptor p75(NTR) (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 p75(NTR) to signal independently of TrkA was investigated with an NGF mutant protein (NGFdelta9/13) that binds poorly to TrkA (Woo et al. [1995] J. Biol. Chem. 270:6278-6285). The NGFdelta9/13 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 NGFdelta9/13 mutant in naive PC12 cells after serum withdrawal, suggesting that p75(NTR) can signal for survival autonomously of TrkA. Annexin V binding, an indication of apoptotic plasma membrane disruption, is inhibited by both NGF and the NGFdelta9/13 mutant after serum deprivation. Both NGF and the NGFdelta9/13 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 NGFdelta9/13 protein and NGF. Finally, upon serum withdrawal, both NGF and the NGFdelta9/13 mutant activate nuclear translocation of the transcriptional factor NF-kappaB (nuclear factor kappaB), a process involved in cell survival. These results are consistent with p75(NTR) inhibition of caspase-mediated apoptosis in PC12 cells. The different physiologic responses elicited by NGFdelta9/13 indicate the potential for individual signaling by the two NGF receptors and also demonstrate the utility of NGF mutants for receptor-selective signal transduction.

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G1/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor supressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases. J. Neurosci. Res. 49:461–474, 1997. © 1997 Wiley-Liss, Inc.

The role of the four histidine residues in receptor binding and activity of mouse nerve growth factor (NGF) was investigated using both site-directed mutagenesis and chemical modification with diethyl pyrocarbonate. Replacement of His-75 or His-84 with alanine resulted in decreased biological activity and decreased affinity for p140(trkA); however, with H75A only, a 5-fold increased affinity toward p75(LANR) was observed. The effect of simultaneous replacement of both His-75 and His-84 was neither additive nor synergistic. Slight perturbations in circular dichroism spectra and weakened self-association of the mutants indicated that His-75 and His-84 may be involved in stability, dimerization, and/or folding of NGF. Diethyl pyrocarbonate modification of His-4 and His-8 in the H75A/H84Q double mutant abolished neuritogenesis, binding to both receptors, and phosphorylation of p140(trkA) in PC12 cells. These chemical and mutational results confirm and clarify previous evidence for the involvement of His-75 and His-84 (Dunbar, J. C., Tregear, G. W., and Bradshaw, R. A. (1984) J. Protein Chem. 3, 349-356) or His-4 and His-8 (Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686) in receptor binding of NGF. At least three and possibly all four histidines, which are located in three spatially distinct regions, contribute to maintenance of functional sites that are essential for receptor binding and activity of NGF.

An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.

The neurotrophins are a family of small dimeric proteins required for the development and survival of vertebrate neurons. Solvent denaturation studies were used to compare recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) to nerve growth factor isolated from mouse submaxillary glands (mNGF). Although greater than 50% sequence identity is conserved among this family, significant structural differences were revealed by the folding and unfolding of these proteins. Denaturation in guanidine hydrochloride and renaturation at pH 7 and 3.5 were monitored by fluorescence intensity, fluorescence polarization, and circular dichroism. The midpoint of equilibrium unfolding curves for all four neurotrophins was independent of the technique but was dependent on protein concentration, indicating that a two-state model involving native neurotrophin dimers and denatured neurotrophin monomers (N2 = 2D) describes the equilibrium between folded and unfolded neurotrophins. The conformational stabilities of the dimeric neurotrophins revealed that mNGF had the lowest conformational stability (19.3 kcal/mol); hNGF, NT-3, and NT-4/5 had intermediate stabilities, and BDNF had the highest stability (26.4 kcal/mol). Recovery of native spectroscopic characteristics upon removal of denaturant indicated that the unfolding process is reversible. Accordingly, unfolding and refolding curves were coincident for mNGF or NT-4/5 at pH 7 and 3.5 and for BDNF at pH 3.5. However, BDNF and NT-3 unfolding and refolding curves were not coincident at pH 7. The stability of the neurotrophins decreased as pH decreased, with compact monomeric intermediates (N2 = [2I] = 2D) becoming populated below pH 4. The differences in stability, pH dependence, and coincidence of refolding curves distinguish the homologous structures of the neurotrophins.

Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse beta-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies--N60, M15, and 27/21--showed any reactivity toward denatured NGF. Three experimental methods--radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots--detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodies to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.

Nerve growth factor (NGF) stimulates rat pheochromocytoma cells (PC12) to differentiate into a neuronal-like cell that exhibits neurite extensions. The role of protein kinase C in signal transduction has been examined in PC12 cells treated with phorbol 12-myristate 13-acetate (PMA) and bryostatin, a macrocyclic lactone that activates protein kinase C at both the nuclear and the plasma membranes [Hocevar, B. A., & Fields, A. P. (1991) J. Biol. Chem. 266, 28-33]. In contrast to PMA down-regulation [Reinhold, D. S., & Neet, K. E. (1989) J. Biol. Chem. 264, 3538-3544], chronic (24 h) treatment with bryostatin blocked the formation of neurites in response to NGF or basic fibroblast-derived growth factor stimulation, but, like PMA, bryostatin did not block the induction of c-fos or c-jun protooncogenes by NGF. Chronic bryostatin treatment down-regulated protein kinase C activity in the cytosolic, membrane, and nuclear fractions. Acute (60 min) bryostatin or NGF treatment activated cytosolic and nuclear protein kinase C activity, suggesting possible translocation to the nucleus. Bryostatin did not induce neurite outgrowth, either alone or in combination with PMA. Thus, the bryostatin-sensitive protein kinase C is distinct from PMA- or K252a-sensitive kinases previously described. The bryostatin-sensitive protein kinase C is necessary, but not sufficient, for neurite outgrowth and acts in the nucleus in a manner independent of c-fos and c-jun transcription.

In the central nervous system, the nerve growth factor (NGF) receptor TrkA is expressed primarily in cholinergic neurons that are implicated in spatial learning and memory, whereas the NGF receptor p75(NTR) is expressed in many neuronal populations and glia. We asked whether selective TrkA activation may have a different impact on learning, short-term memory, and long-term memory. We also asked whether TrkA activation might affect cognition differently in wild-type mice versus mice with cognitive deficits due to transgenic overexpression of mutant amyloid-precursor protein (APP mice). Mice were treated with wild-type NGF (a ligand of TrkA and p75(NTR)) or with selective pharmacological agonists of TrkA that do not bind to p75(NTR). In APP mice, the selective TrkA agonists significantly improved learning and short-term memory. These improvements are associated with a reduction of soluble Aβ levels in the cortex and AKT activation in the cortex and hippocampus. However, this improved phenotype did not translate into improved long-term memory. In normal wild-type mice, none of the treatments affected learning or short-term memory, but a TrkA-selective agonist caused persistent deficits in long-term memory. The deficit in wild-type mice was associated temporally, in the hippocampus, with increased AKT activity, increased brain-derived neurotrophic factor precursor, increased neurotrophin receptor homolog-2 (p75-related protein), and long-term depression. Together, these data indicate that selective TrkA activation affects cognition but does so differently in impaired APP mice versus normal wild-type mice. Understanding mechanisms that govern learning and memory is important for better treatment of cognitive disorders.

Bauhinia purpurea agglutinin (BPA) has been previously characterized as an N-acetylgalactosamine binding protein. The protein has also been demonstrated to possess toric effects on the European corn borer (ECB), a major insect pest of corn. To understand the role of specific amino acid side chins in the biological and biochemical properties of the protein, we have examined the effect of modification of lysine e-NH 2 groups on the conformation and sugar binding properties, as well as the toxicity of BPA toward the corn borer. Modification through guanidination (retention of positive charge) and carbamylation (conversion to neutral residue) indicated some conformational differences between the native and the modified species, although the sugar binding, hemagglutinating, and toric properties were indistinguishable

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage-resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. However, we found no evidence that NK cells produce endogenous proNGF or NGF. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

The processing of polypeptide neurotrophins in the nervous system is poorly understood. In this paper, we provide information on the effects of C-terminal processing of nerve growth factor. Three forms of recombinant mouse beta-nerve growth factor (rNGF) were produced and isolated from insect cells infected with a recombinant baculovirus. The three purified forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with high affinity binding of mouse beta-nerve growth factor (mNGF). However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by gamma-NGF peptidase treatment converted the three forms into a single form identical with mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of gamma-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance.

A comparison has been made of various methods for measuring binding of nerve growth factor (NGF) to PC12 cells in suspension, on plates, and by a combination of the two. Results indicated that the extensive washing in the plate binding assay removed some cell surface ligand, underestimated the fast receptor binding, and overestimated the proportion of internalized ligand. In addition, the binding and internalization by a nonresponding PC12 mutant cell line has been studied. The nonresponding mutants had fewer total NGF receptors (10-50%) than normal cells in any binding assay. However, when measured in the suspension assay, the mutant cells showed both fast and slow binding receptors, in proportion approximately equivalent to those found on native PC12 cells. The PC12 nonresponders in suspension were also found to internalize and degrade low levels of NGF, in proportion to their reduced receptor number. Different results concerning PC12 wild type and mutant cells that have been reported in the literature may be due to the particular binding assay protocol that was used.

We found a pentapeptide conformation, termed a type I twist, which has a strikingly high propensity (56%) for aspartic acid in the first position. Type I twists include the active site loops from cellular and viral aspartic proteases, with the catalytic Asp in the first position. Fifteen other type I twists, from non-homologous proteins, were found among high-resolution structures in the Protein Data Bank using a comparison method based on main-chain torsion angles. We propose that the Asp affects electrostatic interactions and thus plays a major structural role in the formation of this recurring motif, in addition to its catalytic role in the aspartic proteases.

Peripheral sensory neurons are derived from two distinct embryonic tissues, the neural crest and epibranchial placodes. Studies in the chick suggest that embryonic lineage and trophic dependence are interrelated, such that many crest-derived cells depend on NGF for survival during development, whereas placodal derivates, including nodose ganglion neurons, do not (30). It remains controversial, however, whether or not a similar dichotomy exists in mammalian species, in which trophic requirements during early development of placodal ganglia have not been defined. To approach this issue, the present study examined the effects of nerve growth factor (NGF) on neuronal survival in embryonic rat nodose ganglion cultures. Treatment of E13.5–14.5 nodose explants with 20 ng/ml NGF resulted in a four-fold increase in neuronal survival that was blocked by anti-NGF antiserum. Increased neuronal survival and neurite outgrowth were also observed in neuronenriched dissociated cell cultures; these effects were seen within 12 h of plating, indicating that NGF-responsive neurons or neuroblasts were already present in the ganglion at the time of explantation. This was further supported by immunocytochemical staining of nodose cell bodies in situ with the monoclonal antibody 192-IgG against the NGF receptor (12). These findings indicate that NGF may be important in regulating nodose development during early gangliogenesis in mammals and suggest that NGF plays a more widespread role in peripheral nervous system ontogeny than previously recognized.

A slow interconversion between two enzyme forms of rat liver glucokinase has been previously inferred from kinetic assay studies. Two different conformations of the enzyme-substrate complex have now been directly demonstrated by an intrinsic fluorescence enhancement in glucokinase upon addition of glucose. The transition between these two conformations upon glucose addition is measurably slow in the presence of glycerol, with a glucose-dependent half-life of 0.5 to more than 10 min. In the presence of 5% glycerol, the forward and reverse isomerization rate constants are 2.6 x 10(-2) s-1 and 8.5 x 10(-4) s-1, respectively. Correspondingly, the overall equilibrium dissociation constant (0.13 mM) is more than 30-fold lower than the first binding step, i.e. the affinity for glucose is greatly increased. This result was also verified by equilibrium titration of the enzyme with glucose. A similar slow transition was analyzed in the presence of 30% glycerol and observed without glycerol. The dilution of stock glucokinase to promote glucose dissociation from the enzyme showed an exponential fluorescence decay, exactly the reverse phenomenon of glucose addition. The deduced rate constant for the reverse reaction coincided with that calculated from the association results. The conformational change is specific for glucose and responsible for the generation of the kinetic cooperativity of this monomeric enzyme, thus playing a regulatory role in the uptake of glucose in liver.

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both naive and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3′-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for naive and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.

Summary Physiological and pathological aging of the central nervous system (CNS) is characterized by functional neuronal impairments which may lead to perturbed cell homeostasis and eventually to neuronal death. Many toxic events may underlie age-related neurodegeneration. These include the effects of beta amyloid, Tau and mutated presenilin proteins, free radicals and oxidative stress, pro-inflammatory cytokines and lack of growth factor support, which can be individually or collectively involved. Taken individually, these toxicants can induce very diverse cell responses, thus requiring individually targeted corrective interventions upstream of common cell death (apoptotic) pathways. Recent preliminary evidence suggests that the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) and growth factor withdrawal can both activate a common apoptotic pathway in nerve growth factor (NGF)-responsive PC12 cells involving caspase 3, albeit through very distinct upstream pathways: the former through active signalling and the latter through passive or lack of survival signalling. Here, we show that NGF can rescue PC12 cells from both growth factor withdrawal- and TNFα-promoted cell death. However, NGF rescue from growth factor withdrawal requires NGF signalling through the high-affinity tyrosine kinase receptor (TrkA), while NGF rescue from TNFα-promoted cell death requires NGF signalling through the low-affinity p75NTR receptor. These results strengthen the idea that prevention of age- or pathology-associated neurodegeneration may require varied molecular approaches reflecting the diversity of the toxicants involved, possibly acting simultaneously.

A long-standing question in neurotrophin signal transduction is whether heteromeric TrkA-p75NTR complexes possess signaling capabilities that are significantly different from homo-oligomeric TrkA or p75NTR alone. To address this issue, various combinations of transfected PC12 cells expressing a platelet-derived growth factor receptor-TrkA chimera and the p75NTR-selective nerve growth factor mutant (Delta9/13 NGF) were utilized to selectively stimulate TrkA or p75NTR signaling, respectively. The contribution of individual and combined receptor effects was analyzed in terms of downstream signaling and certain end points. The results suggest two unique functions for the high affinity heteromeric NGF receptor site: (a) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth, and (b) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival. Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth in PC12 cells, signaling through p75NTR plays a modulatory role, especially in the increased formation of fine, synaptic "bouton-like" structures, in which both TrkA and p75NTR appear to co-localize. In addition, a new interaction in the TrkA/p75NTR heteromeric receptor signal transduction network was revealed, namely that NGF-induced activation of the MAPK pathway appears to inhibit the parallel NGF-induced Akt pathway.

Alzheimer disease (AD)2 is a debilitating neurodegenerative disorder that directly affects millions of people and indirectly affects the lives of tens of millions of others who must deal with many years of cognitive decline of their loved ones. This devastating disorder, for which no cure is available at present, now strikes someone in America every 71 s. With the current aging population, AD is becoming an ever-increasing social burden for the health care system and national economy in addition to the emotional burden for the immediate family members. AD is pathologically characterized by the presence in the brain of senile plaques containing amyloid-β (Aβ) and neurofibrillary tangles containing Tau. Although these pathological hallmarks were recognized more than a 100 years ago (1–3), only within the past decade have real advances been made in determining the molecular and biochemical basis of AD. This thematic minireview series deals with the current knowledge of the biochemistry of the molecules involved in AD with a view toward understanding the pathobiology of and potential treatments for AD. The first section starts with a discussion of the metabolism and function of the amyloid precursor protein (APP) and plaque formation in AD. The subsequent sections (to be published in later issues) deal with the formation of amyloid oligomers; fibrillization, degradation, and neurotoxicity of Aβ; Tau mutations in AD-like diseases; the association of apoE with AD; and mouse models for AD. The first four minireviews of this thematic series deal with the metabolism and function of APP and how these may affect AD. The first minireview, “Amyloid Precursor Protein Trafficking, Processing, and Function” by Gopal Thinakaran and Edward H. Koo, discusses the biology of APP and its relatives APLP1 and APLP2, with a particular focus on trafficking through the secretory, endocytic, and recycling pathways. The potential function of APP as a trophic, cell adhesion, or receptor molecule is presented with a discussion of the role of APP in the development of the nervous system and synapse structure. The next minireview, by Sarah L. Cole and Robert Vassar, “The Role of Amyloid Precursor Protein Processing by BACE1, the β-Secretase, in Alzheimer Disease Pathophysiology,” initiates a detailed discussion of the processing of APP by BACE1, the enzyme that generates the N terminus of Aβ. The initial identification of the BACE1 enzyme, only 10 years ago, other possible biological roles for BACE1, and the putative development of inhibitors for this ideal target based on its crystal structure round out this review. “Intramembrane Proteolysis by γ-Secretase” by Harald Steiner, Regina Fluhrer, and Christian Haass then develops the story of the elusive nature of the γ-secretase, the enzyme complex made of four subunits, which releases Aβ by cleavage of APP C-terminal fragments. Presenilin, the catalytic subunit, is presented both as an interesting protease that hydrolyzes its substrates in a non-aqueous environment and as the bearer of mutations in familial AD. Of particular interest is the discussion of γ-secretase as a potential target for pharmaceutical intervention and where the prospects currently stand. Phosphorylation is, of course, a main regulatory theme in any biological system, and it certainly holds form for consideration of regulation of APP as detailed in the minireview “Regulation of Amyloid β-Protein Precursor by Phosphorylation and Protein Interactions” by Toshiharu Suzuki and Tadashi Nakaya. The normal function of APP has been hard to pin down, but this minireview carefully outlines the trafficking, transport, and regulation of APP by phosphorylation, with a focus on what the C-terminal fragment may be doing. Taken together, these reviews highlight the known features of APP function, metabolism of APP, the enzymes that cleave it to produce Aβ, and aspects of the physiological regulation of APP processing and Aβ production. The targeting of inhibitors toward certain of these steps to reduce the Aβ load provides optimism for eventual treatment of AD itself. The next section of this series discusses what is believed to be the pathogenic product of APP cleavage, Aβ, in terms of its fibrillization, toxicity, and degradation. In “Structural Classification of Toxic Amyloid Oligomers,” Charles G. Glabe considers various “prefibrillar” forms of Aβ and proposes that conformationally sensitive antibodies might be the best means now for classifying structural types of Aβ oligomers, rather than size. The importance of Aβ degradation as a natural or medicinal means of regulating Aβ levels is discussed in “The AβCs of Aβ-cleaving Proteases” by Malcolm A. Leissring. This minireview evaluates a multitude of Aβ-degrading enzymes that are now known and points to the possibilities of targeting or utilizing this inherent proteolytic activity in the treatment of AD. “Amyloid β-Protein Assembly and Alzheimer Disease” by Robin Roychaudhuri, Mingfeng Yang, Minako Hoshi, and David B. Teplow ranges from a description of the pathway of assembly of Aβ into soluble oligomers and protofibrils to the toxic effects of these assemblies via membrane effects, metals and reactive oxygen species, mitochondrial interactions, and ultimately apoptosis of neurons. Bruce A. Yankner and Tao Lu consider the pathobiological role of Aβ in the minireview “Amyloid β-Protein Toxicity and the Pathogenesis of Alzheimer Disease.” Effects of Aβ oligomers in synaptic physiology, cognitive deficits, Tau interactions, and microglial inflammation lead to novel suggestions for treatments. This 11-part series on AD will be concluded by a discussion of Tau etiology, the important role of apoE in mediating effects of AD, and the contribution of mouse models toward understanding AD pathology. The other side of the Aβ/Tau coin is critically examined in “Tau Mutations in Neurodegenerative Diseases” by Michael S. Wolfe. The role and putative mechanism(s) of aberrant Tau are discussed with an emphasis on splicing of Tau isoforms. The well known genetic association between apoE4 and AD is explored at the molecular level by Ning Zhong and Karl H. Weisgraber in “Understanding the Association of ApoE4 with Alzheimer Disease: Clues from Its Structure.” The emerging information on the structure, stability, domain interactions, and aggregation of apoE isoforms is put in context of neuronal vulnerability, transgenic mice, and AD patients. Finally, no discussion of modern biomedical research would be complete without consideration of genetics and appropriate transgenic mouse models. “Relevance of Transgenic Mouse Models to Human Alzheimer Disease” by Debbi A. Morrissette, Anna Parachikova, Kim N. Green, and Frank M. LaFerla does that very well with a discussion of the advantages and disadvantages of current mouse models and where this approach will move in the future. An understanding of the basic biochemistry of the key players in AD may ultimately provide a framework for developing drugs or other treatments to alleviate the severe pathology these molecules can cause in the elderly. Whether these treatments come as enzyme inhibitors, peptide aggregation blockers, trafficking modulators, lipoprotein mimics, or even Tau antagonists is unknown at this time. We are at a stage in the development of our knowledge of the molecular basis of AD where many people believe that we are poised on the brink of significant breakthroughs; we now need to achieve these next steps before we forget where we are walking.

The effects of a series of protein kinase inhibitors on nerve growth factor (NGF)-dependent and NGF-independent neurite outgrowth in PC12 cells have established an ordered relationship among those protein kinases sensitive to down regulation by bryostatin, stimulation by staurosporine, inhibition by sphingosine, or inhibition by 6-thioguanine (6-TG). Quantitation of the biphasic staurosporine effects on NGF-induced neurite outgrowth (Hashimoto and Hagino: J Neurochem 53:1675-1685, 1989) gave an IC50 of 2-4 nM for inhibition and an EC50 of 15-20 nM for induction of neurite extension. Both sphingosine and 6-TG inhibited neurite outgrowth induced by staurosporine and basic fibroblast derived growth factor (bFGF), as well as by NGF; therefore, sphingosine- and 6-TG-sensitive protein kinase steps occur after the convergence of the NGF, bFGF, and staurosporine signal pathways. Down regulation of protein kinase C by bryostatin chronic treatment, which inhibits NGF- and bFGF-induced neuritogenesis (Singh et al.: Biochemistry 33:542-551, 1994), did not inhibit the staurosporine-induced neurite outgrowth. Thus, the bryostatin-sensitive protein kinase C must occur subsequent to the convergence of the bFGF and NGF pathways, but before (or parallel to) staurosporine initiation of neurite outgrowth. In contrast, low concentrations of phorbol myristoyl acetate (PMA) or bryostatin, which activate protein kinase C activity, enhanced the staurosporine- or NGF-induced neurite extension. These data indicate that stimulation of one or more protein kinase C isozymes can synergistically interact with the signaling pathway to increase the rate of neuritogenesis. Inhibition by 5-7.5 nM staurosporine acted rapidly to arrest and decrease development of neurites up to 24 hr after NGF treatment, as did K252a and NGF polyclonal antibody addition. Our cellular data support the concept that staurosporine acts to inhibit the NGF receptor Trk (Nye et al.: Mol Biol Cell 3:677-686, 1992), but that downstream steps can be activated by the higher concentration of staurosporine to bypass Trk and lead to neurite generation. Effects of staurosporine, 6-TG, and sphingosine on c-fos gene induction with or without NGF were not correlated with the generation of neurites. The sequence of protein kinases sensitive to these effectors appears to be in the order (but not consecutive) bryostatin, staurosporine, sphingosine, and 6-TG.

The role of the NH2-terminal region of nerve growth factor (NGF) was studied with an NGF delta 9/13 deletion mutant, overexpressed in a baculovirus system, and mouse NGF truncated at Met-9 by cleavage with CNBr (des-(1-9)-NGF). Structural studies have been performed on the purified proteins, in addition to biological activity assessment, in order to determine effects of such modifications on global conformation and stability. The activity of NGF delta 9/13 was reduced below detectable levels, and the activity of the des-(1-9)-NGF form was decreased by at least a 50-fold in a PC12 bioassay. Competitive binding of NGF delta 9/13 to low affinity receptors on PC12 cells was not impaired; however, the mutant was not capable of competing for the cold chase-stable, high affinity binding of NGF to the cells. The binding of NGF delta 9/13 to Sf21 cells ectopically expressing the TrkA NGF receptor was also abolished. Thus, deletion of residues 9-13 significantly altered the binding affinity for the high affinity receptors on PC12 cells and for the TrkA receptor, but not for the low affinity receptor. Neither the secondary structure, determined by circular dichroism, nor the conformational stability determined by equilibrium denaturation of NGF delta 9/13 was altered as compared with wild type NGF. Slight conformational and stability perturbations of des-(1-9)-NGF were revealed by the same analysis; however, these changes were found to reflect the influence of the formic acid treatment, not the truncation of 9 residues. Our results support the conclusion that the NH2-terminal domain encompassing residues 1-9 and 9-13 is essential for maintaining the binding capability of NGF for high affinity TrkA receptors. Moreover, conformational and stability data show that the functional results of these modifications of the NH2-terminal region are directly due to receptor binding and not to secondary effects of improper folding or other indirect structural changes.

Publisher Summary This chapter discusses the practical aspects of analyzing and interpreting data— namely, various ways of measuring and quantitating cooperativity, statistical fitting, and evaluation of data and interpretation of such data in terms of simple and complex models that have been proposed to explain the cooperativity of enzymes. The term cooperativity is widely used in biochemical systems to refer to a variety of interactions with a common feature. Cooperativity has been used more recently to describe the association of macromolecular subunits, particularly proteins, into oligomeric structures, in which the initial association of two or more subunits gives rise to an increased affinity, so that succeeding steps in the association occur more readily. The most common usage of the term cooperativity in biochemical processes is with respect to enzyme kinetics and ligand binding to the macromolecules. Cooperativity in ligand binding to a protein can either lead to an enhanced binding of succeeding molecules and a steeper saturation curve than would be expected from non-cooperative binding or lead to a decreased ability of the protein to bind the ligand and a flatter, less-steep saturation curve. It deals primarily with the consequences and causes of spatial interactions on the cooperativity of oligomeric enzymes.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.

The interaction of fluorescein-labeled nerve growth factor (NGF) with human melanoma cells (A875) has been studied in order to assess better methodology for rigorous NGF binding studies. The NGF was modified at a single carboxyl group with iodoacetamidofluorescein after reaction with carbodiimide and cystamine. The modified NGF showed full binding competence in competition with radiolabeled NGF and full biological activity in neurite outgrowth assays compared to native NGF. Binding to unfixed, viable cells was assayed using flow cytometry. This method offers the advantage that unbound ligand need not be separated from that which is cell-associated, thus avoiding pertubation of the binding equilibrium, and accurate, extensive statistical analysis is possible. Binding of fluorescein-NGF was mainly specific and saturable, with analysis by three methods of data treatment indicating a K d of 0.8 to 3 n M at 4°C. Time-based data acquisition allowed a continuous time course for binding to be generated. Binding reached a steady-state level within 5 min of exposure of the cells to the ligand. Kinetic and steady-state results obtained using fluorescein-NGF agree well with previous data produced by 125 I-NGF binding studies. The main limitation of the flow cytometric method in the NGF system is the relative lack of sensitivity compared to the binding of radiolabeled NGF, partially due to unusual quenching of the fluorophore bound to NGF.

Purpose Glaucoma is an optic neuropathy caused by the chronic and progressive death of retinal ganglion cells (RGCs), resulting in irreversible blindness. Ocular hypertension is a major risk factor, but RGC death often continues after ocular hypertension is normalized, and can take place with normal tension. Continuous RGC death was related in rodents and humans to the local upregulation of neurotoxic proteins, such as TNF-α. In rat models of glaucoma, ocular hypertension also upregulates the expression of α2-macroglobulin, which is neurotoxic. α2-macroglobulin upregulation in the retina is long-lived, even after high IOP is reduced with medication. α2-macroglobulin is examined as a possible biomarker in human glaucoma, and a possible neurotoxic mechanism of action is sought. Methods Quantitative Western blotting of α2-macroglobulin in samples obtained from aqueous humor (human and rat) and retina (rat) was conducted. Ex vivo neuronal survival assays and nerve growth factor-α2-macroglobulin binding studies using surface plasmon resonance were used. Results Increased soluble α2-macroglobulin protein is also present in the aqueous humor in a rat glaucoma model, as well as in the aqueous humor of human glaucoma patients but not in cataract patients. One mechanism by which α2-macroglobulin is neurotoxic is by inhibiting the neuroprotective activity of nerve growth factor via TrkA receptors. Conclusions This work further documents a potential novel mechanism of RGC death and a potential biomarker or therapeutic target for glaucoma.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.

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.

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.

Conformationally-linked dissociation equilibria of dimeric proteins have been examined to determine how experimentally obtainable parameters, such as the apparent dissociation constant, k D , and the apparent conformational transition constant, K conf , are related to intrinsic subunit interaction constants, K A or K B , and intrinsic isomerization constants, K 1 or k 2 . Limiting models are considered in which either the conformational change occurs before dissociation or in which the dissociation occurs before the conformational change, as well as a general model including both possibilities. Models are also considered in which three conformations are allowed or in which four subunits (tetramers) are involved. Simulated data for the dimer equilibria are presented to show how variation of protein concentration and variation of certain constants affect the proportion of various molecular species, the weight-average molecular weight, and the overall extent of conformational change. Methods are suggested to distinguish between the different limiting cases based upon the dependence of K D and/or K conf on protein concentration, perturbant concentration, and temperature. It is concluded that methods used to calculate self-dissociation constants oligomeric proteins include linked isomerization reactions such that the equilibrium constant obtained should not be considered as a true subunit interaction term. Indeed, dissociation can occur under the influence of a perturbant with no change in the intrinsic affinity of the subunits but with the sole effect of the perturbant being on a linked conformational change. Additional experiments on the thermodynamics of the conformational change are required to determine the actual relationship. Depending on the complexity of the equilibria involved and the relative value of the equilibrium constants, the extent of the conformational change can vary directly with, vary inversely with, or he independent of the total protein concentration. Even when intrinsic subunit affinities are not affected by the perturbant, the extent of conformational change can vary with protein concentration. Interpretation of data from proteins which may be involved in conformationally-linked dissociation reactions, therefore, must be made with caution.