Abstract
Selenocysteine synthase (SepSecS) catalyzes the terminal reaction of selenocysteine, and is vital for human selenoproteome integrity. Autosomal recessive inheritance of mutations in SepSecS–Ala239Thr, Thr325Ser, Tyr334Cys and Tyr429*–induced severe, early-onset, neurological disorders in distinct human populations. Although harboring different mutant alleles, patients presented remarkably similar phenotypes typified by cerebellar and cerebral atrophy, seizures, irritability, ataxia, and extreme spasticity. However, it has remained unclear how these genetic alterations affected the structure of SepSecS and subsequently elicited the development of a neurological pathology. Herein, our biophysical and structural characterization demonstrates that, with the exception of Tyr429*, pathogenic mutations decrease protein stability and trigger protein misfolding. We propose that the reduced stability and increased propensity towards misfolding are the main causes for the loss of SepSecS activity in afflicted patients, and that these factors contribute to disease progression. We also suggest that misfolding of enzymes regulating protein synthesis should be considered in the diagnosis and study of childhood neurological disorders.
Highlights
We have previously postulated that the A239T variant would bind tRNASec with less affinity compared to wild type (WT) SepSecS, but that its catalytic function would be unaffected[11]
Our results revealed that the pathogenic SepSecS variants–A239T, T325S, and Y334C–are significantly less soluble than the WT enzyme
This analysis is in good agreement with the results of an indirect activity assay in the E. coli system that is based on the substitution of the bacterial selenocysteine synthase with the mutant human SepSecS enzyme
Summary
This subset of patients exhibited a slight reduction in selenoprotein levels, suggesting that SepSecS catalysis was impaired. Studying how point mutations in SEPSECS exert cerebellar dysfunction will shed light onto the role of selenoproteins in the maintenance and development of the human brain. The crystal structure of human SepSecS15 provided a platform for studies on structural and functional effects of these point mutations. The premature stop codon in the Tyr429* variant would result in deletion of elements important for both the integrity of the active site and productive binding of tRNASec 4,11. Our findings suggest that decreased protein stability and the tendency of the SepSecS variants to misfold are the underlying cause of cerebellar atrophy in afflicted patients
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