Molecular Dynamics Calculations of Wild Type vs. Mutant Protein C: Relationship Between Binding Affinity to Endothelial Cell Protein C Receptor and Hereditary Disease

Abstract

Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca²⁺ ion.     

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily,Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266^∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

Crystal structure of a hypothetical protein,TTHA0829 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-β-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four β-strands and two α-helices in a βαββαβ motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.     

Organotypic culture of adult rabbit retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.     

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.     

X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.     

Changes in the thermal dissipation and the electron flow in the water-water cycle in rice grown under conditions of physiologically low temperature

Effects of low temperature on chlorophyll (Chl) fluorescence, gas exchange rate, the amounts of xanthophyll cycle pigments (Xp) and the activities of several antioxidant enzymes were examined in the 8th leaf of two rice (Oryza sativa L.) cultivars (japonica and indica types) and rbcS antisense rice. All plants were grown hydroponically at 25/20 degrees C (day/night), and then exposed to 20/17 degrees C (day/night) after full expansion of the 8th leaf, or exposed to either 20/17 degrees C or 15/13 degrees C (day/night) during the expansion of the 8th leaf. All plants exposed to low temperatures showed a decrease in CO(2) assimilation rate without photoinhibition, and increases in the fraction of thermal dissipation in PSII, and in the electron flux through the water-water cycle (WWC) were observed. Although the increase of thermal dissipation was associated with increases in the ratio of carotenoids to Chl, the ratio of Xp to carotenoids and the de-epoxidation state of Xp, the increase of the electron flux of WWC was not accompanied by an increase in the activities of antioxidant enzymes. Such photoprotective responses did not differ between during and after full expansion of the leaf, and did not differ among the three genotypes. Quantitative analyses on the dissipation of excess light energy showed that thermal dissipation makes a larger contribution than WWC. Thus, although low temperature led to a decrease in CO(2) assimilation, rice potentially coped with the excess light energy by increasing the thermal dissipation and the electron flux of WWC under low temperature irrespective of leaf development and genotypes.     

Effect of irradiance on the partitioning of assimilated carbon during the early phase of grain filling in rice

Low irradiance in the early phase of grain filling in rice often results in a low grain yield, but its effects on the partitioning of previously or recently assimilated carbon within the plant or panicle have not been seriously examined. The objective of this study was to demonstrate the effect of shading during the different stages in the early phase of grain filling on the partitioning of previously or recently assimilated carbon among constituent organs and into superior and inferior spikelets of the panicle in rice (Oryza sativa L. 'Sasanishiki') plants using 13C as a tracer. Plants were grown either under low (shading) or moderate (non-shading) irradiance (120 and 800 micromol quantum m(-2) s(-1)) for 3 or 4 d before or after the 13CO2 feeding at heading, full-heading or milky stages during the early phase of grain filling. Four days after the 13CO2 feeding, the proportion of labelled (previously assimilated) carbon partitioned into the panicle was 17% higher in plants grown under low irradiance compared with plants grown under moderate irradiance at the full-heading stage (7-11 d after heading), while the proportion partitioned into the culm was 13% lower. The light treatments for 3 d were conducted before the 13CO2 feeding and partitioning of the labelled (recently-assimilated) carbon into spikelets was examined 6 h after feeding. The amount of labelled carbon partitioned into the spikelets of the secondary branch (inferior grains) in the plants grown under low irradiance was only 31% when compared with plants grown under moderate irradiance at the full-heading stage, although the partitioning of labelled carbon into the apical spikelets of the primary branch (superior grains) was not affected by the light treatments. These results clearly indicate that preferential partitioning of assimilated carbon into the panicle occurs under low irradiance at around 7-11 d after heading and that the priority of superior spikelets for assimilated carbon intensifies. This phenomenon is thought to be an important strategy for such rice cultivars as used in this study to achieve a certain proportion of ripened grains even under light limited conditions.