Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.     

Recovery of visual functions in a mouse model of Leber congenital amaurosis

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.     

The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco

Summary.  Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using β-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens. Received February 5, 2002; accepted March 27, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, U.S.A.     

Ca(2+) removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca(2+)](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na(+)/Ca(2+) exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-D-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca(2+)](i). In addition, in the absence of extracellular Ca(2+), removal of external Na(+) markedly reduced the rate of loss of Ca(2+) from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na(+) on the rate of Ca(2+) removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca(2+) from the cytosol, while PMCA contributes to the other half of the Ca(2+) removal process.     

Effects of estrogen treatment on glutamate uptake in cultured human astrocytes derived from cortex of Alzheimer's disease patients

Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including the activation of growth factors, the control of synaptic plasticity, and the reduction of response to various insults, such as iron and glutamate. Increasing evidence indicates an increased level of extracellular glutamate and a down-regulation of glutamate transporters in Alzheimer's disease (AD). In this study, we show that glutamate uptake in astrocytes derived from Alzheimer's patients is significantly lower than that from non-demented controls. Estrogen treatment increases glutamate uptake in a dose-dependent pattern. Two glutamate transporters, GLT-1 and GLAST, are expressed in the astrocytes. Up-regulation of the glutamate transporters is induced by estrogen treatment in AD astrocytes only. Our data suggest that the action of estrogen on glutamate uptake by astrocytes might contribute to its potential neuroprotective role in AD.     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

Covariation in the capsid protein of hibiscus chlorotic ringspot virus induced by serial passaging in a host that restricts movement leads to avirulence in its systemic host

Hibiscus chlorotic ringspot virus (HCRSV) from naturally infected Hibiscus rosa-sinensis L. loses virulence in its experimental systemic host Hibiscus cannabinus L. (kenaf) after serial passages in a local lesion host Chenopodium quinoa. Here we report the genetic changes responsible for the loss of virulence at the molecular level. A remarkable covariation of eight site-specific amino acids was found in the HCRSV capsid protein (CP) after serial passages in C. quinoa: Val(49)-->Ile, Ile(95)-->Val, Lys(270)-->Arg, Gly(272)-->Asp, Tyr(274)-->His, Ala(311)-->Asp, Asp(334)-->Ala, and Ala(335)-->Thr. Covariation of at least three of the eight amino acids, Val(49), Ile(95), and Lys(270), caused the virus to become avirulent in kenaf. Interestingly, the nature of the covariation was consistent and reproducible at each serial passage. These data indicate that the nonsynonymous substitutions of amino acids in the HCRSV CP after serial passages in C. quinoa are not likely to be random events but may be due to host-associated positive selection or accelerated genetic drift. The observed interdependence among the three amino acids leading to avirulence in kenaf may have implications for structural or functional relationships in this virus-host interaction.     

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.