The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca 2؉ regulation of guanylate cyclase (GC) by guanylate cyclaseactivating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs؊͞؊ retinas showed no Ca 2؉ dependence, indicating that Ca 2؉ regulation of GCs had indeed been abolished. Flash responses from darkadapted GCAPs؊͞؊ rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs؊͞؊ rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs؊͞؊ rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range.

Five members of a novel Ca2؉ -binding protein subfamily (CaBP), with 46 -58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca 2؉ -binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca 2؉ coordination and the fact that their central ␣-helixes are extended by one ␣-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca 2؉ -mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.Among organisms as diverse as yeast and human, changes in the intracellular Ca 2ϩ ion concentration initiate an array of signaling pathways. Ca 2ϩ ions function as a diffusible signal that exerts its effect directly or through Ca 2ϩ -binding proteins on plasma membrane and intracellular channels, intracellular proteins involved in membrane trafficking, and a broad range of enzymes, including kinases, phosphatases, and adenylyl cyclases. Ca 2ϩ -binding proteins sense changes in [Ca 2ϩ ] through either 130-amino acid (aa) 1 structural elements called C2 domains, 29-aa EF-hand motifs, or through acidic regions of proteins or protein-lipid interfaces. In a growing number of eukaryotic signaling proteins, C2 and EF-hand motifs are present as either a single copy or clustered in multiple copies (1).The largest group of Ca 2ϩ -binding proteins belongs to the calmodulin (CaM) superfamily. They are structurally related and comprise four EF-hand motifs, some of which (one or two) may be nonfunctional in Ca 2ϩ coordination (2). Neuronal Ca 2ϩ -binding proteins (NCBP) are a subset of the EF-hand-containing proteins, whose function is largely unknown. The sequence similarity among members of the NCBP family varies from ϳ25% between CaM and visinin to ϳ60% between GCAP1 and GCAP3 (3). NCBPs are acidic and similar in length. CaM and CaM-like proteins are the shortest (149 -150 aa; molecular mass, 16,837 Da); other members of this family are ϳ200 aa long (molecular mass, ϳ23,000 Da) (2).NCBPs also display a variety of interesting structural features. Multifunctional CaM contains a pair of N-terminal (EFhand 1 and EF-hand 2) and C-terminal EF-hand (EF-hand 3 and EF-ha...

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

The therapeutic imperative for Alzheimer disease (AD) and Parkinson disease (PD) calls for discovery and validation of biomarkers. Increased cerebrospinal fluid (CSF) tau and decreased amyloid (A) beta42 have been validated as biomarkers of AD. In contrast, there is no validated CSF biomarker for PD. We validated our proteomics-discovered multianalyte profile (MAP) in CSF from 95 control subjects, 48 patients with probable AD, and 40 patients with probable PD. An optimal 8-member MAP agreed with expert diagnosis for 90 control subjects (95%), 36 patients with probable AD (75%), and 38 patients with probable PD (95%). This MAP consisted of the following (in decreasing order of contribution): tau, brain-derived neurotrophic factor, interleukin 8, Abeta42, beta2-microglobulin, vitamin D binding protein, apolipoprotein (apo) AII, and apoE. This first large-scale validation of a proteomic-discovered MAP suggests a panel of 8 CSF proteins that are highly effective at identifying PD and moderately effective at identifying AD.

These generally applicable methods establish cutoff values to identify control individuals at increased risk of conversion to mild cognitive impairment which may be useful to people weighing the risk-benefit ratio of new preventive therapeutics and to researchers striving to enrich clinical trial populations with people with latent Alzheimer disease.

In the retinal rod and cone photoreceptors, light photoactivates rhodopsin or cone visual pigments by converting 11-cis-retinal to all-trans-retinal, the process that ultimately results in phototransduction and visual sensation. The production of 11-cis-retinal in adjacent retinal pigment epithelial (RPE) cells is a fundamental process that allows regeneration of the vertebrate visual system. Here, we present evidence that all-trans-retinol is unstable in the presence of H(+) and rearranges to anhydroretinol through a carbocation intermediate, which can be trapped by alcohols to form retro-retinyl ethers. This ability of all-trans-retinol to form a carbocation could be relevant for isomerization. The calculated activation energy of isomerization of all-trans-retinyl carbocation to the 11-cis-isomer was only approximately 18 kcal/mol, as compared to approximately 36 kcal/mol for all-trans-retinol. This activation energy is similar to approximately 17 kcal/mol obtained experimentally for the isomerization reaction in RPE microsomes. Mass spectrometric (MS) analysis of isotopically labeled retinoids showed that isomerization proceeds via alkyl cleavage mechanism, but the product of isomerization depended on the specificity of the retinoid-binding protein(s) as evidenced by the production of 13-cis-retinol in the presence of cellular retinoid-binding protein (CRBP). To test the influence of an electron-withdrawing group on the polyene chain, which would inhibit carbocation formation, 11-fluoro-all-trans-retinol was used in the isomerization assay and was shown to be inactive. Together, these results strengthen the idea that the isomerization reaction is driven by mass action and may occur via carbocation intermediate.

RGS9 is a member of the RGS family of GTPase accelerating proteins (GAPs) for heterotrimeric G proteins. We have explored its contribution to GTPase acceleration in mammalian rod and cone photoreceptors. When RGS9 was specifically removed from detergent extracts of rod outer segments by immunodepletion, the extracts lost nearly all of their GAP activity stimulatable by the inhibitory subunit of cGMP phosphodiesterase. Immunolocalization using monoclonal antibodies and confocal microscopy revealed that RGS9 is present in cones at significantly higher levels than in rods. Thus, RGS9 is the predominant source of GAP activity in rod outer segments, and RGS9 concentration emerges as a potentially important determinant of the faster response kinetics and lower sensitivity of mammalian cones, as compared with rods.

No abstract