RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea

Selenocysteine (Sec), the 21st genetically encoded amino acid, is the major metabolite of the micronutrient selenium. Sec is inserted into nascent proteins in response to a UGA codon. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. While the formation of Sec-tRNASec from seryl-tRNASec by a single bacterial enzyme selenocysteine synthase (SelA) has been well described, the mechanism of Sec-tRNASec formation in archaea and eukaryotes remained poorly understood. Herein, biochemical and genetic data provide evidence that, in contrast to bacteria, eukaryotes and archaea utilize a different route to Sec-tRNASec that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA kinase (PSTK) first converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA: Sec-tRNA synthase (SepSecS); this protein was previously annotated as Soluble Liver Antigen/Liver Pancreas (SLA/LP). SepSecS genes from Homo sapiens, the lower eukaryote Trypanosoma brucei and the archaea Methanocaldococcus jannaschii and Methanococcus maripaludis complement an Escherichia coli DeltaselA deletion strain in vivo. Furthermore, genetic analysis of selenoprotein biosynthesis in T. brucei in vivo demonstrated that eukaryotes have a single pathway to Sec-tRNASec that requires Sep-tRNASec as an intermediate. Finally, purified recombinant SepSecS converts Sep-tRNA Sec into Sec-tRNASec in vitro in the presence of sodium selenite and purified E. coli selenophosphate synthetase.The final step in Sec biosynthesis was further investigated by a structure-based mutational analysis of the M. maripaludis SepSecS and by determining the crystal structure of human SepSecS complexed with tRNA Sec, phosphoserine and thiophosphate at 2.8 A resolution. In vivo and in vitro enzyme assays support a mechanism of Sec-tRNASec formation based on pyridoxal phosphate, while the lack of active site cysteines demonstrates that a perselenide intermediate is not involved in SepSecS-catalyzed Sec formation. Two tRNASec molecules, with a fold distinct from other canonical tRNAs, bind to each human SepSecS tetramer through their unique 13 base-pair acceptor-TPsiC arm. The tRNA binding induces a conformational change in the enzyme\u27s active site that allows a Sep covalently attached to tRNASec, but not free Sep, to be oriented properly for the reaction to occur

Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 Å resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (α2)2 homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process

Ophthalmic Artery Chemosurgery for Less Advanced Intraocular Retinoblastoma: Five Year Review

BACKGROUND: Ophthalmic artery chemosurgery (OAC) for retinoblastoma was introduced by us 5 years ago for advanced intraocular retinoblastoma. Because the success was higher than with existing alternatives and systemic side effects limited we have now treated less advanced intraocular retinoblastoma (Reese-Ellsworth (RE) I-III and International Classification Retinoblastoma (ICRB) B and C). METHODOLOGY/PRINCIPAL FINDINGS: Retrospective review of 5 year experience in eyes with Reese Ellsworth (Table 1) I (7 eyes), II (6 eyes) or III (6 eyes) and/or International Classification (Table 2) B (19 eyes) and C (11 eyes) treated with OAC (melphalan with or without topotecan) introduced directly into the ophthalmic artery. Patient survival was 100%. Ocular event-free survival was 100% for Reese-Ellsworth Groups I, II and III (and 96% for ICRB B and C) at a median of 16 months follow-up. One ICRB Group C (Reese-Ellsworth Vb) eye could not be treated on the second attempt for technical reasons and was therefore enucleated. No patient required a port and only one patient required transfusion of blood products. The electroretinogram (ERG) was unchanged or improved in 14/19 eyes. CONCLUSIONS/SIGNIFICANCE: Ophthalmic artery chemosurgery for retinoblastoma that was Reese-Ellsworth I, II and III (or International Classification B or C) was associated with high success (100% of treatable eyes were retained) and limited toxicity with results that equal or exceed conventional therapy with less toxicity

Outcomes of Descemet Stripping Endothelial Keratoplasty Using Eye Bank-Prepared Preloaded Grafts

To evaluate the feasibility of Descemet stripping endothelial keratoplasty using grafts preloaded by an eye bank in a commercially available insertion device. In this retrospective case series, a series of 35 eyes in 34 consecutive patients who underwent Descemet stripping endothelial keratoplasty for Fuchs endothelial dystrophy or previously failed full-thickness grafts at a single tertiary care center from March 2013 to March 2014 was included. The donor tissue had undergone pre-lamellar dissection, trephination, and loading into EndoGlide Ultrathin inserters at the Lions Eye Institute for Transplant and Research (Tampa, FL) and was shipped overnight in Optisol GS to the surgeon (K.C.). Surgery was performed within 24 hours from tissue preparation and loading by the eye bank. Donor and recipient characteristics, endothelial cell density (ECD), best-corrected visual acuity, and central corneal thickness were recorded. The main outcome measures were intraoperative and postoperative complications and ECD loss at 3, 6, and 12 months. One primary graft failure (2.8%), 2 rebubblings (5.7%), and 1 graft rejection (2.8%) occurred. Mean preoperative donor ECD was 2821 ± 199 cells/mm. Six months postoperatively, the mean endothelial cell loss was 25.3% ± 17.2% (n = 32), which remained stable at 1 year (31.5% ± 17.9%, n = 32). Mean best-corrected visual acuity improved from 20/100 preoperatively to 20/25 at a mean follow-up of 1 year (n = 32). Mean central corneal thickness was reduced from 711 ± 110 μm to 638 ± 66 μm at the last follow-up visit. Donor graft tissue preloaded by an eye bank can be used successfully for endothelial keratoplasty. Preloading reduces intraoperative tissue manipulation

Corneal neovascularization in childhood keratitis

Introduction: Cornea clarity is essential for optimal vision at any age. In childhood, loss of corneal transparency may also lead to amblyopia. Thus, proper management of the conditions that lead to corneal scarring and neovascularization in children is of utmost importance. Areas covered: Herein, we review the pathophysiology of the main causes of corneal inflammation, scarring and neovascularization in childhood. A review of the literature was performed using the keywords 'herpetic keratitis', 'blepharokeratoconjunctivitis', 'ocular rosacea', 'phlyctenular conjunctivitis', 'vernal keratoconjunctivitis', and 'corneal neovascularization' in combination with the words 'children' or 'childhood'. Expert commentary: Regardless of the underlying cause of the inflammatory stimulus - viral infection, meibomian gland secretions, atopy - scarring and neovascularization occur when expression of pro-angiogenic factors outweighs that of anti-angiogenic ones. Proper control of the inflammation will restore the equilibrium between pro- and anti-angiogenic enzymes and cytokines and, in turn, limit the resulting scarring and neovessel formation. Early diagnosis and therapy for herpetic infection, blepharokeratoconjunctivitis, and atopic disease can preserve and/or restore corneal clarity in childhood keratitis