A d-Amino Acid-Containing Neuropeptide Discovery Funnel.

A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.

Characterization of GdFFD, a D-amino acid-containing neuropeptide that functions as an extrinsic modulator of the Aplysia feeding circuit.

During eukaryotic translation, peptides/proteins are created using L-amino acids. However, a D-amino acid-containing peptide (DAACP) can be produced through post-translational modification via an isomerase enzyme. General approaches to identify novel DAACPs and investigate their function, particularly in specific neural circuits, are lacking. This is primarily due to the difficulty in characterizing this modification and due to the limited information on neural circuits in most species. We describe a multipronged approach to overcome these limitations using the sea slug Aplysia californica. Based on bioinformatics and homology to known DAACPs in the land snail Achatina fulica, we targeted two predicted peptides in Aplysia, GFFD, similar to achatin-I (GdFAD versus GFAD, where dF stands for D-phenylalanine), and YAEFLa, identical to fulyal (YdAEFLa versus YAEFLa), using stereoselective analytical methods, i.e. MALDI MS fragmentation analysis and LC-MS/MS. Although YAEFLa in Aplysia was detected only in an all L-form, we found that both GFFD and GdFFD were present in the Aplysia CNS. In situ hybridization and immunolabeling of GFFD/GdFFD-positive neurons and fibers suggested that GFFD/GdFFD might act as an extrinsic modulator of the feeding circuit. Consistent with this hypothesis, we found that GdFFD induced robust activity in the feeding circuit and elicited egestive motor patterns. In contrast, the peptide consisting of all L-amino acids, GFFD, was not bioactive. Our data indicate that the modification of an L-amino acid-containing neuropeptide to a DAACP is essential for peptide bioactivity in a motor circuit, and thus it provides a functional significance to this modification.

Quantitative cone contrast threshold testing in patients with differing pathophysiological mechanisms causing retinal diseases.

BACKGROUND: Cone contrast threshold testing (CCT) provides quantitative measurements of color and contrast function to reveal changes in vision quality that are not standard endpoints in clinical trials. We utilize CCT to measure visual function in patients with multiple sclerosis (MS), age-related macular degeneration (AMD), epiretinal membrane (ERM), and retinal vein occlusion (RVO). METHODS: Retrospective data was gathered from 237 patients of the Gavin Herbert Eye Institute. Subjects included 17 patients with MS, 45 patients with AMD, 41 patients with ERM, 11 patients with RVO, and 123 healthy controls. Patients underwent the primary measurement outcome, CCT testing, as well as Sloan visual acuity test and spectral domain optical coherence tomography during normal care. RESULTS: Color and contrast deficits were present in MS patients regardless of history of optic neuritis. AMD with intermediate or worse disease demonstrated reduced CCT scores. All 3 stages of ERM demonstrated cone contrast deficits. Despite restoration of visual acuity, RVO-affected eyes demonstrated poorer CCT performance than unaffected fellow eyes. CONCLUSIONS: CCT demonstrates color and contrast deficits for multiple retinal diseases with differing pathophysiology. Further prospective studies of CCT in other disease states and with larger samples sizes is warranted.

An Atypical Presentation of Propionibacterium Acnes Endophthalmitis Following Cataract Surgery.

Purpose: To describe a case with an unusual presentation of Propionibacterium acnes (P acnes) with ultimately a good visual outcome. Methods: A case report with review of approaches to P acnes endophthalmitis. Results: We describe a patient with an unusual presentation of P acnes of panuveitis with white, circular preretinal lesions without intracapsular deposits. Diagnosis was made from cultures from pars plana vitrectomy. Eventually, she was definitively managed with capsulectomy, repositioning of her intraocular lens via sutureless intrascleral fixation, and intravitreal vancomycin injection. Conclusion: This is a report of P acnes endophthalmitis presenting with discrete preretinal lesions where surgical and medical management lead to a complete resolution of uveitis and symptoms after a 3-year follow up where the patient's final visual acuity was Snellen 20/20 OU.

TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1.

Certain proteins can undergo polyglycylation and polyglutamylation. Polyglutamylases (glutamate ligases) have recently been identified in a family of tubulin tyrosine ligase-like (TTLL) proteins. However, no polyglycylase (glycine ligase) has yet been reported. Here we identify a polyglycylase in the TTLL proteins by using an anti-poly-glycine antibody. The antibody reacted with a cytoplasmic 60-kDa protein that accumulated in elongating spermatids. Using tandem mass spectrometry of trypsinized samples, immunoprecipitated by the antibody from the TTLL10-expressing cells, we identified the 60-kDa protein as nucleosome assembly protein 1 (NAP1). Recombinant TTLL10 incorporated glycine into recombinant NAP1 in vitro. Mutational analyses indicated that Glu residues at 359 and 360 in the C-terminal part of NAP1 are putative sites for the modification. Thus, TTLL10 is a polyglycylase for NAP1.

Aplysia Locomotion: Network and Behavioral Actions of GdFFD, a D-Amino Acid-Containing Neuropeptide.

One emerging principle is that neuromodulators, such as neuropeptides, regulate multiple behaviors, particularly motivated behaviors, e.g., feeding and locomotion. However, how neuromodulators act on multiple neural networks to exert their actions remains poorly understood. These actions depend on the chemical form of the peptide, e.g., an alternation of L- to D-form of an amino acid can endow the peptide with bioactivity, as is the case for the Aplysia peptide GdFFD (where dF indicates D-phenylalanine). GdFFD has been shown to act as an extrinsic neuromodulator in the feeding network, while the all L-amino acid form, GFFD, was not bioactive. Given that both GdFFD/GFFD are also present in pedal neurons that mediate locomotion, we sought to determine whether they impact locomotion. We first examined effects of both peptides on isolated ganglia, and monitored fictive programs using the parapedal commissural nerve (PPCN). Indeed, GdFFD was bioactive and GFFD was not. GdFFD increased the frequency with which neural activity was observed in the PPCN. In part, there was an increase in bursting spiking activity that resembled fictive locomotion. Additionally, there was significant activity between bursts. To determine how the peptide-induced activity in the isolated CNS is translated into behavior, we recorded animal movements, and developed a computer program to automatically track the animal and calculate the path of movement and velocity of locomotion. We found that GdFFD significantly reduced locomotion and induced a foot curl. These data suggest that the increase in PPCN activity observed in the isolated CNS during GdFFD application corresponds to a reduction, rather than an increase, in locomotion. In contrast, GFFD had no effect. Thus, our study suggests that GdFFD may act as an intrinsic neuromodulator in the Aplysia locomotor network. More generally, our study indicates that physiological and behavioral analyses should be combined to evaluate peptide actions.

Structure, bioactivity, and resistance mechanism of streptomonomicin, an unusual lasso Peptide from an understudied halophilic actinomycete.

Natural products are the most historically significant source of compounds for drug development. However, unacceptably high rates of compound rediscovery associated with large-scale screening of common microbial producers have resulted in the abandonment of many natural product drug discovery efforts, despite the increasing prevalence of clinically problematic antibiotic resistance. Screening of underexplored taxa represents one strategy to avoid rediscovery. Herein we report the discovery, isolation, and structural elucidation of streptomonomicin (STM), an antibiotic lasso peptide from Streptomonospora alba, and report the genome for its producing organism. STM-resistant clones of Bacillus anthracis harbor mutations to walR, the gene encoding a response regulator for the only known widely distributed and essential two-component signal transduction system in Firmicutes. To the best of our knowledge, Streptomonospora had been hitherto biosynthetically and genetically uncharacterized, with STM being the first reported compound from the genus. Our results demonstrate that understudied microbes remain fruitful reservoirs for the rapid discovery of novel, bioactive natural products.

Estradiol-17beta stimulates specific receptor and endogenous nitric oxide-dependent dynamic endothelial protein S-nitrosylation: analysis of endothelial nitrosyl-proteome.

Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.