Selective conservation of GAP-43 structure in vertebrate evolution

GAP-43 (a.k.a. B-50, F1, pp46, or neuromodulin) is a major growth cone membrane protein whose expression is widely correlated with successful axon elongation, but whose function remains unknown. To distinguish the structural features of GAP-43 most relevant to its cellular functions, we have determined features of the protein that are most highly conserved in vertebrate evolution. Comparison of fish and mammalian GAP-43 distinguishes two domains of the protein. A strictly conserved amino-terminal domain contains the putative site for fatty acylation and membrane attachment, a calmodulin binding domain, and a proposed phosphorylation site. In the much larger carboxy-terminal domain, amino acid composition is strongly conserved without extensive sequence conservation. This amino acid composition predicts an extended, negatively charged rod conformation with some similarity to the side arms of neurofilaments. The results suggest that the biological roles of GAP-43 may depend on an ability to form a dynamic membrane-cytoskeleton-calmodulin complex.     

Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43

Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP-43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent-enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.     

Association of an axonally transported polypeptide (H) with 100-A filaments. Use of immunoaffinity electron microscope grids

Polypeptide H (mol wt 195,000) is axonally transported in rabbit retinal ganglion cells at a velocity of 0.7--1.1 mm/d, i.e., in the most slowly moving of the five transport groups described in these neurons. To identify the organelle with which H is associated, we purified H, prepared antibodies directed against it, and adsorbed the antibodies onto Formvar-coated electron microscope grids. When the resulting "immuno-affinity grids" were incubated with extracts of spinal cord and then examined in the electron microscope, they contained as many as 100 times more 100-A filaments than did grids coated similarly with nonimmune IgG. The ability of the anti-H IgG to specifically adsorb filaments to grids was completely blocked by incubating the IgG with polypeptide H. The 100-A filaments adsorbed to anti-H immunoaffinity grids could be specifically decorated by incubating them with anti-H IgG. These observations demonstrate that H antigens (and most likely H itself) are associated with 100-A neurofilaments. In addition, they suggest that the use of immunoaffinity grids may be a useful approach for determining the organelle associations of polypeptides.     

The Shift-Work Accident Rate is More Related to the Shift Type than to Shift Rotation

The current study investigated the accident rates across morning, late, and night shifts in rotating shift-workers employed in two different shift rotations at the same steel work factory. A retrospective analysis has been performed of accident data (N = 578) over a 5-year period (2003 through 2007) of 730 male shift-workers employed in either a clockwise (mean age of the workers 38.1 ± SD 9.8 years) or counterclockwise rotation (mean age 38.0 ± SD 10.1 years) with comparable work conditions. The overall accident rate across the 24-h day was not significantly different between clockwise and counterclockwise shift rotation. In both shift-work rotations, morning shifts as opposed to night shifts exhibited a significantly higher accident rate. There was no significant difference between late shifts and morning or night shifts in either shift rotation. The increased accident rate in the morning shift at this steel factory could be related to the early starting time of the shift and to this shift being more labor intensive in both shift rotations. These findings suggest that work-related factors must be considered in addition to shift-work schedules when investigating accident rates in rotating shift-workers.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

Improved tests for heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

The neutral theory of molecular evolution predicts that the ratio of polymorphisms to fixed differences should be fairly uniform across a region of DNA sequence. Significant heterogeneity in this ratio can indicate the effects of balancing selection, selective sweeps, mildly deleterious mutations, or background selection. Comparing an observed heterogeneity statistic with simulations of the heterogeneity resulting from random phylogenetic and sampling variation provides a test of the statistical significance of the observed pattern. When simulated data sets containing heterogeneity in the polymorphism-to-divergence ratio are examined, different statistics are most powerful for detecting different patterns of heterogeneity. The number of runs is most powerful for detecting patterns containing several peaks of polymorphism; the Kolmogorov-Smirnov statistic is most powerful for detecting patterns in which one end of the gene has high polymorphism and the other end has low polymorphism; and a newly developed statistic, the mean sliding G statistic, is most powerful for detecting patterns containing one or two peaks of polymorphism with reduced polymorphism on either side. Nine out of 27 genes from the Drosophila melanogaster subgroup exhibit heterogeneity that is significant under at least one of these three tests, with five of the nine remaining significant after a correction for multiple comparisons, suggesting that detectable evidence for the effects of some kind of selection is fairly common.      

Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tRNA synthetase

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.     

Light-induced melatonin suppression in humans with polychromatic and monochromatic light

The relative contribution of rods, cones, and melanopsin to non-image-forming (NIF) responses under light conditions differing in irradiance, duration, and spectral composition remains to be determined in humans. NIF responses to a polychromatic light source may be very different to that predicted from the published human action spectra data, which have utilized narrow band monochromatic light and demonstrated short wavelength sensitivity. To test the hypothesis that only melanopsin is driving NIF responses in humans, monochromatic blue light (lambda(max) 479 nm) was matched with polychromatic white light for total melanopsin-stimulating photons at three light intensities. The ability of these light conditions to suppress nocturnal melatonin production was assessed. A within-subject crossover design was used to investigate the suppressive effect of nocturnal light on melatonin production in a group of diurnally active young male subjects aged 18-35 yrs (24.9+/-3.8 yrs; mean+/-SD; n=11). A 30 min light pulse, individually timed to occur on the rising phase of the melatonin rhythm, was administered between 23:30 and 01:30 h. Regularly timed blood samples were taken for measurement of plasma melatonin. Repeated measures two-way ANOVA, with irradiance and light condition as factors, was used for statistical analysis (n=9 analyzed). There was a significant effect of both light intensity (p<0.001) and light condition (p<0.01). Polychromatic light was more effective at suppressing nocturnal melatonin than monochromatic blue light matched for melanopsin stimulation, implying that the melatonin suppression response is not solely driven by melanopsin. The findings suggest a stimulatory effect of the additional wavelengths of light present in the polychromatic light, which could be mediated via the stimulation of cone photopigments and/or melanopsin regeneration. The results of this study may be relevant to designing the spectral composition of polychromatic lights for use in the home and workplace, as well as in the treatment of circadian rhythm disorders.