Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3

The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded b-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)3 RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the b-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family

トクシマ ダイガク ビョウイン ニュウイン カンジャ ノ シカテキ ニーズ : コウクウ カンリ センター ニオケル ウケイレ ジョウキョウ カラ

Tokushima University Hospital Oral Health Management Center was created in April 2006 in order that medical inpatients could be easily referred to the dental section. From April 2007 to August 2009, among the 658 inpatients referred by the Oral Health Management Center, 210 requested professional oral healthcare. Requests for professional oral healthcare are currently increasing. Among 244 patients who asked for a bedside visit, 184 requested professional oral healthcare. Most inpatients were referred from the Division of Blood Internal Medicine; and the second Division referring numerous inpatients was that of Neurological Disorders. The increased tendency for professional oral healthcare requests might be related to the closer working relations among NST staff in the hospital, and/or better knowledge regarding the importance of oral health by medical doctors and nurses. The needs for dental services for inpatients varies. It is important for dental professionals to cooperate with medical care staff, and to be capable of dealing with various general conditions

UVA ユウドウ ヒフ ヒカリロウカ ニオケル シシツ カサンカブツ ノ ヤクワリ ト コウサンカ ブッシツ セッシュ ノ エイキョウ

Role of ultraviolet A（UVA）on oxidative damage has attracted much attention in relation to skin photoaging process. In this study, we investigated effect of cholesterol hydroperoxides（Chol- OOHs）, stable products of reactive oxygen species（ROS）-induced lipid peroxidation, on matrix metalloproteinase（MMP）activation responsible for wrinkle formation in hairless mouse skin and then estimated the inhibition of UVA-induced MMP activation by dietary β-carotene. Hairless mice were exposed to UVA irradiation for 8 weeks. Chol-OOHs content in the skin was found to increase significantly by the exposure of UVA. In addition, the activity of MMP-9 and its protein expression were elevated with wrinkle formation. This activation was also induced by intracutaneous injection of Chol-OOHs. Interestingly, dietary β-carotene（500 mg/kg diet） and α-tocopherol （100 mg/kg diet）suppressed the accumulation of Chol-OOH as well as MMP activation. These results suggest that Chol-OOHs formed by the exposure of UVA to skin contribute to the activation of MMPs resulting in skin photoaging. Dietary antioxidants may prevent skin photoaging through, at least partly, the suppression of MMP activation due to UVA-induced lipid peroxidation

Identification and Modulation of the Key Amino Acid Residue Responsible for the pH Sensitivity of Neoculin, a Taste-Modifying Protein

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular

Structural basis for the dual RNA-recognition modes of human Tra2-beta RRM

Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mTOR pathway. Treatment with rapamycin, an mTOR inhibitor, or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach

Attorney SHINKAI Takahiro, KAWAMURA Yoshisada, YOKOYAMA Akira

神大法曹会の人々弁護士 新開崇弘・川村宜禎・横山朗さんに聞

CNVs in Three Psychiatric Disorders

BACKGROUND: We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). METHODS: Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. RESULTS: In genic CNVs, we found an increased burden of smaller (500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25–0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. CONCLUSIONS: BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD

在来種ネズミモチと移入種トウネズミモチ(モクセイ科) の保全遺伝学的研究

Genetic introgression from introduced, non-native species into native populations is a growing challenge for biological conservation, and one that raises unique practical and ethical issues. Ligustrum lucidum is native to China, and cultivated or used in gardens in various areas in Japan. Recently, some studies reported that this species escaped from cultivated areas and was sympatric with L. japonicum. This indicates that L. japonicum faces the ecological and genetic risk of hybridization and introgression with non-native L. lucidum. Therefore, we examined whether hybridization between L. japonicum and the non-native L. lucidum has occurred in a coexisting population using phenological and molecular analyses. The phenological results indicate that there is very little overlap in the flowering times of the two species. Moreover, molecular analyses using PCR-RFLP of chloroplast and nuclear DNA sequences could not detect any hybridization or introgression of L. lucidum and L. japonicum in the population.移入種から在来種への遺伝子移入は生物学的保全のための懸念課題であり，実用的かつ倫理的な問題を提起している。中国原産のトウネズミモチ（Ligustrum lucidum）は日本の広い地域で園芸として栽植されているが逸出により分布を拡大しており，在来種のネズミモチ（L. japonicum）と同所的に生育していることが近年報告されている。このため，ネズミモチはトウネズミモチとの雑種形成や浸透交雑の生態学的リスクにさらされていると考えられる。そこで本研究では，在来種のネズミモチと移入種のトウネズミモチの間での雑種形成による遺伝的攪乱の有無を明らかにすることを目的として，開花期調査および分子遺伝学的調査を行った。開花期の調査結果より，両種の開花期がずれていることが明らかとなった。PCR-RFLP解析の結果，ネズミモチとトウネズミモチの間に交雑個体および浸透交雑個体を検出することはできなかったため，両種間での交雑は起こっていないと考えられる