SummaryIn a search for Proteus mirabilis genes that were regulated by cell-to-cell signalling, a lacZ fusion ( cmr437:: mini-Tn 5lacZ ) was identified that was repressed 10-fold by a self-produced extracellular signal from wild-type cells. However, the cmr437 ::miniTn 5lacZ insertion itself led to a marked reduction in this extracellular repressing signal. The cmr437 ::miniTn 5lacZ insertion was mapped to a speA homologue in P. mirabilis. Sequence analysis indicated that a speB homologue was encoded downstream of speA . Products of the SpeA and SpeB enzymes (agmatine and putrescine) were tested for repression of cmr437::lacZ . Agmatine did not have repressing activity. However, putrescine was an effective repressing molecule at concentrations down to 30 m m m m M. A second prominent phenotype of the cmr437 ( speA )::miniTn 5lacZ insertion was a severe defect in swarming motility. This swarming defect was also observed in a strain containing a disruption of the downstream speB gene. Differentiation of the speB mutant to swarmer cells was delayed by two hours relative to wild-type cells. Furthermore, the speB mutant was unable to migrate effectively across agar surfaces and formed very closely spaced swarming rings. Exogenous putrescine restored both the normal timing of swarmer cell differentiation and the ability to migrate to speB mutants.

,f,i and the Clinical Genomic and Proteomic AMD Study Group Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by ϳ60 and ϳ30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2-3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ϳ76% accuracy and in combination with genomic markers provide up to ϳ80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease. Molecular & Cellular Proteomics 8:1338 -1349, 2009. Age-related macular degeneration (AMD)1 is the most common cause of legal blindness in the elderly in developed countries (1). It is a complex, progressive disease involving multiple genetic and environmental factors that can result in severe visual loss. Early risk factors include the macular deposition of debris (drusen) on Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE). Later stages of "dry" AMD involve the degeneration of photoreceptor and RPE cells resulting in geographic atrophy. In "wet" AMD, abnormal blood vessels grow from the choriocapillaris through Bruch membrane (choroidal neovascularization (CNV)). CNV occurs in 10 -15% of AMD cases yet accounts for over 80% of debilitating visual loss in AMD. Anti-vascular endothelial growth factor treatments can effectively inhibit the progression of CNV (1), and antioxidant vitamins and zinc can slow dry AMD progression for select individuals (2). However, there a...

Epidermal growth factor receptor (EGFr) is a key mediator of cell communication during animal development and homeostasis. In Drosophila, the signaling event is commonly regulated by the polytopic membrane protein Rhomboid (RHO), which mediates the proteolytic activation of EGFr ligands, allowing the secretion of the active signal. Until very recently, the biochemical function of RHO had remained elusive. It is now believed that Drosophila RHO is the founder member of a previously undescribed family of serine proteases, and that it could be directly responsible for the unusual, intramembranous cleavage of EGFr ligands. Here we show that the function of RHO is conserved in Gram-negative bacteria. AarA, a Providencia stuartii RHO-related protein, is active in Drosophila on the fly EGFr ligands. Vice versa, Drosophila RHO-1 can effectively rescue the bacterium's ability to produce or release the signal that activates density-dependent gene regulation (or quorum sensing). This study provides the first evidence that prokaryotic and eukaryotic RHOs could have a conserved role in cell communication and that their biochemical properties could be more similar than previously anticipated.

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4-to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.Cell-to-cell communication in bacteria is mediated by a wide variety of distinct chemical signals, and a number of reviews on this subject have been compiled (11,12,17,22). In gramnegative bacteria, these signals include the N-acylhomoserine lactones, autoinducer 2 (AI-2), quinolones, cyclic dipeptides, and the tryptophan derivative indole (2,12,26,32,38). In gram-positive bacteria, peptides are the primary mode of communication (11,17,22). Although peptide-mediated signals have not been isolated from gram-negative bacteria, recent data suggest that such systems are present (13,29,30).Several extracellular signals have been identified in Escherichia coli. Indole, a product of the tryptophanase-mediated catabolism of tryptophan, is capable of activating the astD, tnaB, and gabT genes (38). Studies using a tnaA mutant unable to produce indole have indicated that a second signal is required for full activation of the astD and gabT genes (38). A second signal in E. coli, the AI-2 furanone signal, has been shown by microarray analysis to control the expression of a large number of genes in E. coli (8,34). Additional genes in E. coli that are regulated by undefined extracellular signals include rpoS, sdiA, cysK, and ftsQAZ (1,14,23,33,37).The cysteine regulon in E. coli comprises a set of genes involved in the biosynthesis of cysteine from L-serine (18,19). These genes are positively controlled by the CysB activator in concert with the molecule N-acetyl-L-serine (NAS) (19). The production of NAS occurs in two steps. First, se...

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case/control cohort. We tested the top findings for replication in 1 896 cases and 1 866 controls and identified two novel genetic protective factors for AMD. In addition to the CFH (p=2.3×10−64) and ARMS2 (p=1.2×10−60) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (p=5.3×10−15), a gene near the C2/BF locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (p=2.9×10−4), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

. Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration (Prph Rd2/ϩ ), which was crossed onto a Nyx nob background to eliminate the b-wave and most other postreceptoral ERG components. On this background, Prph Rd2/ϩ mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in Prph Rd2/ϩ mice than predicted by rod photoreceptor activity (Rm P3 ) or anatomical analysis. At 4 mo of age, the RPE in Prph Rd2/ϩ mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease.

The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the ␤-lactam antibiotic cefotaxime.

Toward early detection of susceptibility to age-related macular degeneration (AMD), we quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. Mean CEP adduct and autoantibody levels were elevated in AMD plasma by ∼60 and ∼30%, respectively, and the odds ratio for both CEP markers elevated was ∼3-fold greater in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high-temperature requirement factor A1 (HTRA1), complement factor H (CFH), and complement C3. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2- to 3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ∼76% accuracy and in combination with genomic markers, provide up to ∼80% discrimination accuracy. CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for predicting susceptibility to this blinding disease.