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Abstract: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. We identified 7 novel and 2 previously reported mutations in six T2-deficient patients. Transient expression analysis of wild-type and eight mutant cDNAs was performed at 40, 37 and 30°C. Although no significant residual activity was detected, mutant proteins were detected in the N158D, N158S, R208Q, Y219H and N282H mutants. Accumulation of these mutant proteins was temperature-sensitive with the highest expression levels at lower temperatures. Expression of Q73P and N353K cDNAs yielded neither residual T2 protein nor enzyme activity. An E252del mutant T2 was detected with a relative protein amount and enzyme activity of 30% and 25%, respectively, in comparison to the wild-type at 37°C. The E252del mutant protein was more stable at 30°C expression than 37°C, but was essentially undetectable at 40°C, indicating its temperature-sensitive instability. Kinetic studies revealed a twofold K m elevation for substrates coenzyme A and acetoacetyl-CoA in the E252del mutant, while V max was comparable to the wild-type. We conclude that the E252del is a temperature-sensitive K m mutant. This correlates well with the effect predicted from the T2 tertiary structure analysis, using the crystal structure of the human T2 homotetramer. The probable effect of the other mutations on the T2 tertiary structure was also evaluated. [Copyright &y& Elsevier]

Context: Pegpesen® is a novel Y-shape pegylated recombinant human growth hormone (rhGH) for once-weekly treatment of children with growth hormone deficiency (GHD)., Objective: To evaluate the efficacy and safety of Pegpesen® in children with GHD vs daily rhGH., Design: A multicenter, randomized, controlled phase 3 clinical trial with a duration of 52 weeks treatment., Setting: 23 centers in China., Participants: 391 pediatric participants diagnosed with GHD., Interventions: Participants were randomized 2:1 to the weekly Pegpesen® group (140 μg/kg/week) or the daily rhGH group (245 μg/kg/week) for 52 weeks., Main Outcome Measures: The primary endpoint was the growth velocity (GV) at 52 weeks, and the secondary endpoints mainly involved changes from baseline in height standard deviation scores for chronological age and bone age (ΔHt SDS CA and ΔHt SDS BA)., Results: At 52 weeks, the least squares mean (LS Means) of GV was 9.910 cm/year in the Pegpesen® group and 10.037 cm/year in the daily rhGH group. The LS Means difference between groups was -0.127 [95% CI (-0.4868, 0.2332)], confirming that weekly Pegpesen® is non-inferior to daily rhGH. The LS Means of ΔHt SDS CA, ΔHt SDS BA, were similar across both groups (all P > 0.05). Safety profiles and adherence were comparable., Conclusions: Pegpesen® was non-inferior to daily rhGH, with similar safety, lower dosage requirements, thus presenting a new therapeutic option for children with GHD., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

The survival of motor neuron (SMN) protein forms a multiprotein complex (SMN complex) with Gemin proteins. The complex is known to play a crucial role in RNA metabolism. Several lines of evidence show that SMN is phosphorylated at serine and/or threonine residues. In this study, we hypothesized that SMN is phosphorylated at two kinds of serine residues, the Q28SDD31SD site and two SQ sites (80SQ and 163SQ). A FLAG-tagged wild-type construct (SMNfull) and three FLAG-tagged mutant constructs were made: an SMNAQ mutant with two AQ sites instead of two SQ sites at residues 80 and 163, an SMNQADDAD mutant with QADDAD instead of Q28SDD31SD, and an SMNAQ/QADDAD mutant with the two AQ sites and QADDAD. We expressed these mutants in HeLa cells and analyzed their phosphorylated bands by immunoblotting, the protein stability using cycloheximide, binding to Gemin 2 and foci formation. Mutations in Q28SDD31SD, but not in two SQ sites reduced the intensity of phosphorylation bands, indicating that Q28SDD31SD is the major phosphorylation site in SMN. Mutations in the two SQ sites and Q28SDD31SD did not affect protein stability and binding to Gemin 2. Whereas mutations in the two SQ sites did not cause apparent changes in foci formation, mutations in Q28SDD31SD resulted in a reduced number of large foci in the cytosol. We demonstrated that phosphorylation in Q28SDD31SD may be important in cytosolic foci formation.