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Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) (OMIM #617481) is an autosomal recessive disease characterized by progressive microcephaly, plagiocephaly, hypotonia, spastic quadriparesis, global developmental delay, intellectual disability, optic features and abnormal brain magnetic resonance imaging (MRI). NMIHBA was recently reported to be caused by PRUNE1 mutations. Eight mutations have been reported in 13 unrelated families. Here, we report 3 PRUNE1 mutations in 1 Caucasian and 3 Japanese families. One recurrent missense mutation (p.Asp106Asn) was previously reported in Turkish and Italian families, while the other 2 mutations (p.Leu18Serfs*8 and p.Cys180*) are novel. We also show that mutant PRUNE1 mRNA can be subject to nonsense-mediated mRNA decay. The patients presented in this study showed atypical NMIHBA phenotypes with no progressive microcephaly. Furthermore, one Caucasian case had significant macrocephaly; therefore, patients with PRUNE1 mutations can exhibit a broad and heterogeneous spectrum of phenotypes., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The vanadate (Vi)-mediated photocleavage reaction was used to study the interaction between the regulatory segment and the catalytic site of smooth muscle myosin light chain kinase (MLCK). When MLCK was irradiated with long-wave UV (366 nm) in the presence of ADP and Vi, kinase activity was substantially decreased, and the MLCK polypeptide of 130 kDa was cleaved into several smaller fragments with apparent molecular masses of 100, 70, 60, 32, and 28 kDa. Inhibition of kinase activity and photocleavage were both competitively antagonized by the addition of ATP. Inconsistency between the observed maximum levels of UV-induced inhibition of MLCK-mediated phosphorylation (80%) and photocleavage (15-20%) suggested that the photocleavage reaction proceeds as a two-step process. Monoclonal antibodies recognizing the C-terminus of MLCK labeled the 60- and 28-kDa fragments, indicating that MLCK was cleaved at two sites, at 28 and 60 kDa from the C-terminus, within what are believed to be the autoinhibitory region and the catalytic site, respectively. Moreover, Ca2+-calmodulin (Ca2+-CaM) protected against cleavage at the site at 28 kDa from the C-terminus. Analysis of the amino acid composition of the fragment revealed that the cleavage site at 28 kDa from C-terminus occurred at Lys 799 +/- 3 amino acid residues, which is in a region where the CaM-binding and pseudosubstrate regions overlap. These results suggest that the three-dimensional structure of MLCK brings the regulatory segment into direct contact with the ATP-binding site. Moreover, the binding of Ca2+-CaM displaces the regulatory segment away from the catalytic site.