Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA^Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N^ε-acetyl-Lys (AcK) onto tRNA^Pyl. Here, we examine an N^ε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.The standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with site-specifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10–12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15–19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell growth revealed, over 50 y ago, that many ncAAs were incorporated into proteins by the regular translation machinery (10). Thus, it was not surprising to see that many of the successful orthogonal Methanococcus jannaschii tyrosyl-tRNA synthetase variants (20) facilitate incorporation of multiple different ncAAs (21–23). This polyspecificity is also a property of the orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pair (reviewed in ref. 24).PylRS variants that facilitate site-specific insertion of N^ε-acetyl-l-Lys (AcK; 2) (Fig. 1A) into proteins were derived from directed evolution experiments (25–28). These AcK-tRNA synthetase (AcKRS) enzymes have been used to investigate the role of acetylation sites in tumor suppressor p53 (29) and histone H3 (30). Here, we present biochemical and structural studies showing that AcKRS variants are polyspecific and catalytically deficient enzymes compared with canonical AARSs. These AcKRSs selected by directed evolution to ligate AcK to tRNA^Pyl are actually ∼30-fold more efficient in activation with Phe derivatives. Crystallographic structures of AcKRS and PylRS variants in complex with AcK, 3-iodo-l-Phe (3-I-Phe; 4) (Fig. 1A), or 2-(5-bromothienyl)-l-Ala (3-Br-ThA; 10) (Fig. 1A) reveal the structural basis of polyspecificity in these engineered PylRS enzymes.Open in a separate windowFig. 1.(A) Chemical structures of nsAAs used in the study: 1, l-pyrrolysine (Pyl); 2, AcK; 3, N^ε-trifluoroacetyl-l-Lys (CF₃-AcK); 4, 3-I-Phe; 5, 3-bromo-l-Phe (3-Br-Phe); 6, 3-chloro-l-Phe (3-Cl-Phe); 7, 3-trifluoromethyl-l-Phe (3-CF₃-Phe); 8, 3-methyl-l-Phe (3-Me-Phe); 9, 3-methoxyl-l-Phe (3-MeO-Phe); 10, 3-Br-ThA. (B) Range of substrate specificity of AcKRS3. Translation of the sfGFP reporter (UAG codon at position 2) by the library of ncAA-tRNA^Pyl was measured by fluorescence intensity. A library of 94 different Lys and Phe analogs (Dataset S1) was tested. Fluorescence signals from the incorporation of ncAAs 2–5, 7, 9, and 10 are labeled in A. Well A12 is a control without added ncAA. Well A1 was a positive control experiment to detect the production signal of WT sfGFP (100%). Fluorescence data and error values (SD) are represented by bars from three independent experiments (also given in Dataset S1). Colors (0–4% in purple, 4–8% in blue, 18–24% in red, and 95–100% in brown) have been used to indicate the level of UAG read-through.     

Feasibility of an accelerated PVAL method for the collection of GSR and biological traces

International Journal of Legal Medicine - The polyvinyl alcohol method (PVAL) is known as an effective technique to thoroughly collect traces of gunshot residue (GSR) from different surfaces, e.g.,...     

Subnormothermic machine perfusion for preservation of porcine kidneys in a donation after circulatory death model

Machine perfusion for preservation led to compelling success for the outcome of renal transplantation. Further refinements of methods to decrease preservation injury remain an issue of high interest. This study investigates functional and morphological aspects of kidneys preserved by subnormothermic (20 °C) machine perfusion (SNTM) compared with oxygenated hypothermic machine perfusion (HMPox) and cold storage (CS) in a donation after circulatory death (DCD) model. After 30 min of warm ischaemia, porcine kidneys were randomly assigned to preservation for 7 h by CS, HMPox or SNTM. Afterwards, kidneys were reperfused for 2 h with autologous blood in vitro for assessment of function and integrity. Application of SNTM for preservation led to significantly higher blood flow and urine output compared with both other groups. SNTM led to a twofold increased creatinine clearance compared with HMPox and 10‐fold increased creatinine clearance compared with CS. Structural integrity was best preserved by SNTM. In conclusion, this is the first study on SNTM for kidneys from DCD donors. SNTM seems to be a promising preservation method with the potential to improve functional parameters of kidneys during reperfusion.     

MELD at POD 1 as a predictor of outcome in liver allografts with peak AST >5000 U/l

Perioperative liver graft injury is associated with elevation of aminotransferases after orthotopic liver transplantation (OLT). Values above 5000 U/l usually are regarded as extreme liver graft injury (ELGI). Some patients and organs recover from this critical condition. The aim of the study was to evaluate factors contributing to graft and patient survival after ELGI. From chart review we identified 64 of 917 OLT adult patients (median age 54.2 years; 68.8% males) transplanted between 11/2003 and 02/2012, who presented ELGI after OLT. Donor and recipient factors were analyzed and correlated with the outcome by univariable and multivariable methods. Multivariable cox proportional hazards showed that recipient's BMI (P = 0.01), model for end stage liver disease (MELD) score before OLT (P = 0.02) and laboratory MELD score 24 h after OLT (P = 0.01) were independently associated with patient survival. 30‐days and 12‐months survival in patients with a postoperative laboratory MELD higher than 31 was 21.4%, while patients with a postoperative laboratory MELD lower than 31 displayed 30‐days and 12‐months survival rates of 80% and 71.8%, respectively (P < 0.001). Retransplantation in the setting of ELGI after OLT should be based on all available data. Utilization of the postoperative labMELD enables the transplant physician within 24 h after transplantation to identify necessity of retransplantation objectively.     

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative

Objectives

Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials.

Aims

This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.

Materials and methods

EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining.

Results

Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points.

Conclusion

The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings.

Clinical relevance

The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.