Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome)

Early infantile epileptic encephalopathy with suppression-burst pattern (EIEE) is one of the most severe and earliest forms of epilepsy, often evolving into West syndrome; however, the pathogenesis of EIEE remains unclear. ARX is a crucial gene for the development of interneurons in the fetal brain, and a polyalanine expansion mutation of ARX causes mental retardation and seizures, including those of West syndrome, in males. We screened the ARX mutation and found a hemizygous, de novo, 33-bp duplication in exon 2, 298_330dupGCGGCA(GCG)9, in two of three unrelated male patients with EIEE. This mutation is thought to expand the original 16 alanine residues to 27 alanine residues (A110_A111insAAAAAAAAAAA) in the first polyalanine tract of the ARX protein. Although EIEE is mainly associated with brain malformations, ARX is the first gene found to be responsible for idiopathic EIEE. Our observation that EIEE had a longer expansion of the polyalanine tract than is seen in West syndrome is consistent with the findings of earlier onset and more-severe phenotypes in EIEE than in West syndrome.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

ent-Eudesmane sesquiterpenoids, galloyl esters of the oak lactone precursor, and a 3-O-methylellagic acid glycoside from the wood of Platycarya strobilacea

ent-Eudesmane sesquiterpenoids, 8,11-dihydroxy-2,4-cycloeudesmane, 11-hydroxy-2,4-cycloeudesman-8-one and 2,4-cyclo-7(11)-eudesmen-8-one, were isolated from the wood of Platycarya strobilacea, which has been used as an aromatic tree since at least the 18th century. On charring the wood, 2,4-cyclo-7(11)-eudesmen-8-one was detected in the smoke. In the charred wood, the concentrations of ellagitannins, such as galloyl pedunculagin, dramatically decreased, whereas concentrations of pentagalloyl glucose, and other gallotannins were relatively stable. In addition, two other compounds, the 6′-O-m- and p-digalloyl oak lactone precursor and the 3-O-methylellagic acid 4′-O-(4″-O-galloyl)-xylopyranoside, were isolated from the charred wood along with m- and p-digallic acid.     

Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP).

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages.     

Production of novel antioxidative prenyl naphthalen-ols by combinational bioconversion with dioxygenase PhnA1A2A3A4 and prenyltransferase NphB or SCO7190

We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.     

CpG site degeneration triggered by the loss of functional constraint created a highly polymorphic macaque drug-metabolizing gene, CYP1A2

Background

Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation) and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD) event (ohnologs) versus small-scale duplications (SSD) to determine if there exist any differences in their patterns of sequence evolution.

Results

For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like) in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression.

Conclusions

Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.     

Molecular Characterization of an Intact p53 Pathway Subtype in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.