Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.     

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C₁ and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7^Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.     

Carbonic anhydrase inhibitors. Benzenesulfonamides incorporating cyanoacrylamide moieties strongly inhibit Saccharomyces cerevisiae β-carbonic anhydrase

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogs) were assayed as inhibitors of the β-carbonic anhydrase (CA, EC 4.2.1.1) from Saccharomyces cerevisiae, ScCA. Some of these compounds were low nanomolar or subnanomolar ScCA inhibitors and showed selectivity ratios in the range of 4.91–69.86 for inhibiting the yeast enzyme over the offtarget human (h) isoforms hCA I and of 6.46–13.52 for inhibiting ScCA over hCA II. The model organism S. cerevisiae and this particular enzyme may be useful for detecting antifungals with a novel mechanism of action compared to the classical azole drugs to which significant drug resistance emerged. Indeed, some of these sulfonamides inhibited the growth of the yeast with CC₅₀-s in the range of 0.73–6.54 μM.     

An evaluation of sequencing coverage and genotyping strategies to assess neutral and adaptive diversity

Whole genome sequences (WGS) greatly increase our ability to precisely infer population genetic parameters, demographic processes, and selection signatures. However, WGS may still be not affordable for a representative number of individuals/populations. In this context, our goal was to assess the efficiency of several SNP genotyping strategies by testing their ability to accurately estimate parameters describing neutral diversity and to detect signatures of selection. We analysed 110 WGS at 12× coverage for four different species, i.e., sheep, goats and their wild counterparts. From these data we generated 946 data sets corresponding to random panels of 1K to 5M variants, commercial SNP chips and exome capture, for sample sizes of five to 48 individuals. We also extracted low‐coverage genome resequencing of 1×, 2× and 5× by randomly subsampling reads from the 12× resequencing data. Globally, 5K to 10K random variants were enough for an accurate estimation of genome diversity. Conversely, commercial panels and exome capture displayed strong ascertainment biases. Besides the characterization of neutral diversity, the detection of the signature of selection and the accurate estimation of linkage disequilibrium (LD) required high‐density panels of at least 1M variants. Finally, genotype likelihoods increased the quality of variant calling from low coverage resequencing but proportions of incorrect genotypes remained substantial, especially for heterozygote sites. Whole genome resequencing coverage of at least 5× appeared to be necessary for accurate assessment of genomic variations. These results have implications for studies seeking to deploy low‐density SNP collections or genome scans across genetically diverse populations/species showing similar genetic characteristics and patterns of LD decay for a wide variety of purposes.     

Iron-superoxide dismutase and monodehydroascorbate reductase transcripts accumulate in response to internode rubbing in tomato

A cDNA encoding an iron-superoxide dismutase (Fe-SOD) was isolated by RACE-PCR from a Lycopersicon esculentum cDNA library. The Fe-SOD cDNA consists of a 746-bp open reading frame and is predicted to encode a protein of 249 amino acids with a calculated molecular mass of 27.9 kDa. The deduced amino acid sequence was very similar to other plant Fe-SODs and a potential chloroplastic targeting was found. To study the induction of oxidative burst in response to mechanical stimulation, the accumulation of Fe-SOD and monodehydroascorbate reductase (MDHAR) mRNAs was analysed in response to young growing internode rubbing in tomato plants. Northern analyses show that Fe-SOD mRNA and MDHAR mRNA accumulated in tomato internodes 10 min after the mechanical stimulation. These results suggest that reactive oxygen species are early involved in the response of a plant to a mechanical stimulation, such as rubbing. The nucleotide sequence data reported in this paper will appear in the NCBI Nucleotide Sequence Databases under the accession number AY262025.     

Sequential protein association with nascent 60S ribosomal particles

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.