Characterization of yeast seryl-tRNA synthetase active site mutants with improved discrimination against substrate analogues.

The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding was demonstrated previously [B. Lenhard et al., J. Biol. Chem. 272 (1997) 1136-1141]. In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, two new active site mutants, SerRS11 and SerRS12, were constructed. The catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases were tested. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (Lys287Thr, Asp288Tyr, Ala289Val) and SerRS12 (Lys287Arg) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate, an analogue of serine, and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine, a stable analogue of aminoacyl adenylate, than the wild-type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in the motif 2 loop.

Combined MRI lesions and relapses as a surrogate for disability in multiple sclerosis.

OBJECTIVE: In multiple sclerosis (MS), the aim of therapies is to prevent the accumulation of irreversible disability. This is difficult to assess given the short time course of clinical trials. MRI markers and relapses are often used as surrogate of disability in MS studies, but their validity remains controversial. We sought to validate, at the individual patient level, MRI lesions and relapses as surrogates for disability progression over the course of MS trials. METHODS: Individual patient data from a large, placebo-controlled trial of interferon ß-1a in relapsing-remitting MS (RRMS) were analyzed. The Prentice criteria were applied to evaluate surrogacy of 1-year MRI active lesions and relapses for disability worsening (Expanded Disability Status Scale [EDSS]) over the 2-year follow-up. RESULTS: All Prentice criteria were satisfied. Treatment reduced by 31% the odds of having EDSS worsening over 2 years, reducing the mean number of MRI lesions by 61% and the mean number of relapses by 36% over 1 year. Both 1-year MRI lesion activity and relapses, when considered independently, accounted for more than 60% of the treatment effect on 2-year EDSS worsening. A combination of 1-year MRI lesion activity and relapses explained 100% of the treatment effect on EDSS worsening over 2 years. CONCLUSIONS: A combined measure of 1-year changes in MRI lesions and relapses after interferon therapy fully estimated the corresponding effect on 2-year EDSS worsening. This short-term combined measure appears to be a surrogate for disability progression over a longer term when evaluating the effect of interferon in RRMS.