However, we found that the kinase activity of LRRK2 is important for stabilizing cellular levels of LRRK2 because an artificial variant of LRRK2 lacking kinase activity was markedly and consistently destabilized even in the absence of exogenous CHIP (Figure 3C).
To address the function of PINK1 in living cells, we generated stable dopaminergic neuroblastoma lines that express wild-type PINK1, a recessive mutant, G309D and an artificial variant lacking kinase activity [29] (Fig. 1A C).
Biochemists have been harnessing an artificial version of evolution to refine natural enzymes, making new variants that work faster, longer, and at higher temperatures.
To test whether polyubiquitination is required for CHIP-mediated LRRK2 degradation, we co-transfected HEK293 cells with an artificial ubiquitin variant, UbK0, in which all lysines were replaced by arginines to prevent polyubiquitination [39].
Sequencing technology-specific error signatures [ 17] can yield artificial variant calls of erroneous high quality and result in an underestimated proportion of false calls, while poorly adjusted bioinformatics pipelines for data processing may lead to missed calls.
Various peptide acceptor substrates were used in the present study: a peptide based on a part of the sequence of bikunin (Bik, QEEEGSGGGGQR), two variants of a sequence based on amino acids 55 65 from Drosophila syndecan (Syn, DDDSIEGSGGR, and the artificial variant Syn2, DDDSIEGSGSGGR, which has two Ser-Gly motifs) and a sequence based on part of human perlecan (Perl, DSISGDDLGSGDLGSGDFQR).
For example, in a proof of concept study, known mutations and artificial variants in exon18 BRCA1 were introduced using the CRISPR/Cas9 technique, resulting in traceable changes in BRCA1 mRNA splicing and abundance [ 43].
Cloning and expression of native genes and synthetic artificial variants of the consensus sequence repeats from the native genes has been accomplished.
In addition, several artificial variants were designed to target putative regulatory motifs: c.8484A > T and c.8486A > C, c.8512T > A, c.8518del and two combined BIC variants c.[8609A > G 8611G > T] and c.[8972G > A; 9006A > T] within the same construction.
Similar effects on Km have been shown for artificial variants of the homologous arginine residue R387 of the ACAD isovaleryl-CoA dehydrogenase (32) and have been predicted from crystal structure analysis for the ACAD glutaryl-CoA dehydrogenase (33).
To ascertain the nature of these splicing anomalies two artificial variants were designed and generated: c.8484A > T (intact splice site and SF2 disruption) and c.8486A > C (weak alteration of splice site: 0.95 →0.92 and SF2 disruption; Additional file 1, Table S1).
