Edward Ki Yun Leung

The Mechanism of Peptidyl Transfer Catalysis by the Ribosome

The ribosome catalyzes two fundamental biological reactions: peptidyl transfer, the formation of a peptide bond during protein synthesis, and peptidyl hydrolysis, the release of the complete protein from the peptidyl tRNA upon completion of translation. The ribosome is able to utilize and distinguish the two different nucleophiles for each reaction, the α-amine of the incoming aminoacyl tRNA versus the water molecule. The correct binding of substrates induces structural rearrangements of ribosomal active-site residues and the substrates themselves, resulting in an orientation suitable for catalysis. In addition, active-site residues appear to provide further assistance by ordering active-site water molecules and providing an electrostatic environment via a hydrogen network that stabilizes the reaction intermediates and possibly shuttles protons. Major questions remain concerning the timing, components, and mechanism of the proton transfer steps. This review summarizes the recent progress in structural, biochemical, and computational advances and presents the current mechanistic models for these two reactions.

Clinical evaluation of the QMS® Tacrolimus immunoassay.

BACKGROUND Tacrolimus, a widely used immunosuppressant, inhibits T-lymphocyte signal transduction and cytokine upregulation. We evaluated and compared the performance of a newly developed tacrolimus immunoassay method to LC-MS/MS. METHOD Analytical performance was assessed using quality control materials and whole blood patient samples. Interferences studies were performed using pooled whole-blood samples spiked with each interferent, respectively. Comparison studies were conducted using 145 de-identified whole blood samples collected after routine tacrolimus analysis by LC-MS/MS. RESULTS CVs were between 3.9 and 8.1% and the method was linear (r(2)=0.99) up to 30.0 ng/ml. Calibration was stable ≤12 days and LOQ was 0.7 ng/ml (14.4% CV). Bilirubin (≤48 mg/dl), hemoglobin (≤345 mg/dl), and triglycerides (<2800 mg/dl) showed no significant interference. Comparison (Passing-Bablok regression) for all samples showed a proportional bias of 17%. Comparisons of liver and kidney transplant patients showed slope biases of 22% and 31%, respectively, whereas other remaining transplant patients (stem cell, heart, lung, and islet) showed a slope bias of 0.98. CONCLUSIONS Overall, the QMS Tacrolimus Immunoassay showed good analytical performance. Comparison studies showed a proportional bias of 17%, which can be attributed to the significant number of liver and kidney transplant patients present in this study (121/145).

Reducing the risk of hyperammonemia from transfusion of stored red blood cells

Ammonia concentration increases in red cell units (RBCs) during storage. We measured absolute amounts of ammonia (AA) per unit serially in stored RBCs and before and after removal of the supernatant by volume reduction (VR) or washing. Ammonia increased 6.4-fold in untreated units over 31 days. VR decreased AA 3.7-fold, whereas washing decreased it 38-fold (p<0.0001). At least for certain patients, e.g., infants receiving large volume transfusions and patients in liver failure, it may be advisable to use RBCs as fresh as possible and to limit infusion (by VR or washing) of ammonia in the supernatant.

Transition State Features in the Hepatitis Delta Virus Ribozyme Reaction Revealed by Atomic Perturbations.

Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single-strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the hepatitis delta virus (HDV) ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double-mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group.

Development and validation of a targeted affinity-enrichment and LC–MS/MS proteomics approach for the therapeutic monitoring of adalimumab

BACKGROUND The anti-tumor necrosis factor alpha (TNFα) therapeutic monoclonal antibodies (mAbs), such as adalimumab, are widely used in the treatment of rheumatoid arthritis, inflammatory bowel diseases, and other auto-immune diseases. The administration of adalimumab can elicit the immune responses from some patients, resulting in the formation of anti-drug antibodies (ADAbs). The ADAbs can diminish the therapeutic effects of adalimumab by neutralizing the TNFα binding site or increasing its clearance from circulation. METHODS To effectively monitor the therapeutic concentrations of adalimumab, we developed and validated a targeted quantitative proteomic assay to determine the circulating concentrations of adalimumab. Since drug effects can be attenuated by ADAbs, the method adopted an affinity-enrichment step to selectively quantify the bioavailable forms of adalimumab in patient serum samples. RESULTS The performance of the LC-MS/MS based assay provides the analytical measuring range and precisions applicable for the therapeutic monitoring of adalimumab. It also provides comparable results to a cell-based activity assay when evaluating patient samples with different concentrations of adalimumab. CONCLUSION Our assay can quantify both sub-therapeutic and therapeutic concentrations of bioavailable adalimumab in patient serum samples. This assay design provides an alternative to isotope-labeled peptides approach currently adopted in targeted proteomics methods.

Smith-Lemli-Opitz Syndrome in a newborn infant with developmental abnormalities and low endogenous cholesterol

BACKGROUND Patients with Smith-Lemli-Opitz Syndrome (SLOS) have defective endogenous cholesterol synthesis, and present with decreased cholesterol levels and multiple developmental dysmorphologies. CASE DESCRIPTION A newborn infant with normal XY karyotype and normal microarray was born with multiple developmental defects and ambiguous genitalia. The patient was diagnosed with SLOS, following biochemical genetic analysis of serum 7-DHC concentrations. The clinical course of the patient was further complicated by the comorbidities associated with SLOS and the bacterial infections. CONCLUSION We provide a detailed biochemical profile of the SLOS patient. The report can help us further understand the pathological impacts of cholesterol synthesis deficiency and provide relevant clinical management with outcome of this rare genetic disorder.

Diiodothyropropionic acid (DITPA) cross-reacts with thyroid function assays on different immunoassay platforms

Monocarboxylate transporter 8 (MCT8) is a thyroid hormonespecific cell membrane transporter that is expressed in numerous human tissues, including the brain, skeletal muscle, heart, lung, and kidney [1]. Loss-of-function mutations to the MTC8 gene, located on chromosomeXq13.2, producing severe psychomotor deficits and anunusual combination of thyroid function test abnormalities were identified in 2004 [2,3]. A year later it was demonstrated that subjects with Allan–Herndon–Dudley syndrome with X-chromosome linked mental retardation [4] also had the same thyroid hormone test abnormalities and mutations in the MCT8 gene [5]. The defects manifested in boys at infancy are hypotonia and poor head control. The neurodevelopmental defect progresses to result in spastic quadriplegia and inability to walk and talk [2,3,5]. Affected individuals have high serum triiodothyronine (TT3), very low reverse T3 (rT3), slight reduction in thyroxine (TT4) and low or mildly elevated thyrotropin (TSH) [2,3,5]. Genetically engineered MCT8 deficient mice demonstrated that the absence of MCT8 produces thyroid hormone deprivation in the brain and the T3 excess results in hypermetabolism in peripheral tissue explaining the combination of tissue specific hypothyroidism (brain) and hypermetabolism leading to the inability to gain weight [6]. A thyroid hormone analogue, diiodothyropropionic acid (DITPA) was found to enter cells independently of MTC8 [7]. Therefore, this compound was tested in children with MCT8 deficiency and found to normalize their thyroid function tests, reduce the T3-induced hypermetabolism and, thus, improve their nutritional status [8]. A problem in the follow-up of DITPA treatment is the cross-reactivity of DITPA in the routine laboratory measurements of T3 (total and free). This necessitates themeasurement of DITPA by LC–MS/MS to correct for its interference in T3 determination by immunoassay. However, the degree of cross-reactivity of DITPA with TT3 assays on different platforms has not been explored. To investigate this, we studied the possible cross-reactivity of DITPA on commercial thyroid hormone immunoassays available from four in-vitro diagnostic companies.