Molecules

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12 pages, 3162 KiB

Open AccessArticle

Pro-Oxidant Activity of an ALS-Linked SOD1 Mutant in Zn-Deficient Form

by Chise Nagao, Kunisato Kuroi, Taiyu Wakabayashi and Takakazu Nakabayashi

Molecules 2020, 25(16), 3600; https://doi.org/10.3390/molecules25163600 - 07 Aug 2020

Cited by 3 | Viewed by 2683

Cu, Zn superoxide dismutase (SOD1) is a representative antioxidant enzyme that catalyzes dismutation of reactive oxygen species in cells. However, (E,E)-SOD1 mutants in which both copper and zinc ions were deleted exhibit pro-oxidant activity, contrary to their antioxidant nature, at physiological temperatures, following [...] Read more.

Cu, Zn superoxide dismutase (SOD1) is a representative antioxidant enzyme that catalyzes dismutation of reactive oxygen species in cells. However, (E,E)-SOD1 mutants in which both copper and zinc ions were deleted exhibit pro-oxidant activity, contrary to their antioxidant nature, at physiological temperatures, following denaturation and subsequent recombination of Cu²⁺. This oxidative property is likely related to the pathogenesis of amyotrophic lateral sclerosis (ALS); however, the mechanism by which Cu²⁺ re-binds to the denatured (E,E)-SOD1 has not been elucidated, since the concentration of free copper ions in cells is almost zero. In this study, we prepared the (Cu,E) form in which only a zinc ion was deleted using ALS-linked mutant H43R (His43→Arg) and found that (Cu,E)-H43R showed an increase in the pro-oxidant activity even at physiological temperature. The increase in the pro-oxidant activity of (Cu,E)-H43R was also observed in solution mimicking intracellular environment and at high temperature. These results suggest that the zinc-deficient (Cu,E) form can contribute to oxidative stress in cells, and that the formation of (E,E)-SOD1 together with the subsequent Cu²⁺ rebinding is not necessary for the acquisition of the pro-oxidant activity. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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12 pages, 2079 KiB

Open AccessArticle

Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection

by Takuya Terai, Tomoyuki Koike and Naoto Nemoto

Molecules 2020, 25(6), 1472; https://doi.org/10.3390/molecules25061472 - 24 Mar 2020

Cited by 3 | Viewed by 4223

Abstract

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially [...] Read more.

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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18 pages, 738 KiB

Open AccessArticle

Nanoscale Quantum Thermal Conductance at Water Interface: Green’s Function Approach Based on One-Dimensional Phonon Model

by Toshihito Umegaki and Shigenori Tanaka

Molecules 2020, 25(5), 1185; https://doi.org/10.3390/molecules25051185 - 05 Mar 2020

Cited by 2 | Viewed by 2453

Abstract

We have derived the fundamental formula of phonon transport in water for the evaluation of quantum thermal conductance by using a one-dimensional phonon model based on the nonequilibrium Green’s function method. In our model, phonons are excited as quantum waves from the left [...] Read more.

We have derived the fundamental formula of phonon transport in water for the evaluation of quantum thermal conductance by using a one-dimensional phonon model based on the nonequilibrium Green’s function method. In our model, phonons are excited as quantum waves from the left or right reservoir and propagate from left to right of H

_{2}

O layer or vice versa. We have assumed these reservoirs as being of periodic structures, whereas we can also model the H

_{2}

O sandwiched between these reservoirs as having aperiodic structures of liquid containing N water molecules. We have extracted the dispersion curves from the experimental absorption spectra of the OH stretching and intermolecular modes of water molecules, and calculated phonon transmission function and quantum thermal conductance. In addition, we have simplified the formulation of the transmission function by employing a case of one water molecule (N=1). From this calculation, we have obtained the characteristic that the transmission probability is almost unity at the frequency bands of acoustic and optical modes, and the transmission probability vanishes by the phonon attenuation reflecting the quantum tunnel effect outside the bands of these two modes. The classical limit of the thermal conductance calculated by our formula agreed with the literature value (order of

10^{- 10}

W/K) in high temperature regime (>300 K). The present approach is powerful enough to be applicable to molecular systems containing proteins as well, and to evaluate their thermal conductive characteristics. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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13 pages, 1798 KiB

Open AccessArticle

Fluorescence Differentiation of ATP-Related Multiple Enzymatic Activities in Synovial Fluid as a Marker of Calcium Pyrophosphate Deposition Disease Using Kyoto Green

by Nattha Yongwattana, Nutsara Mekjinda, Tulyapruek Tawonsawatruk, Itaru Hamachi, Akio Ojida and Jirarut Wongkongkatep

Molecules 2020, 25(5), 1116; https://doi.org/10.3390/molecules25051116 - 02 Mar 2020

Cited by 2 | Viewed by 4154

Abstract

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of [...] Read more.

Calcium pyrophosphate deposition disease (CPPD) is a crystal induced inflammation in joints, and causes severe pain in elderly people. The accumulation of pyrophosphate (PPi) in synovial fluid (SF) results from several enzymatic reactions, especially the highly activated e-NPPs, which catalyze the conversion of ATP to PPi. This study demonstrates the detection of relative catalytic activity of 3 enzymes—ecto-nucleotide pyrophosphatase/phosphodiesterases (e-NPPs), tissue nonspecific alkaline phosphatase (TNAP), and ecto-nucleoside triphosphate diphosphohydrolases (e-NTPDases)—using a single molecular sensor called Kyoto Green. Kyoto Green exhibits excellent performance in sensing the catalytic activity of the commercial representatives of the e-NPPs, TNAP, and e-NTPDases, which are ENPP1, PPase, and apyrase, respectively, in both single-enzyme and multi-enzyme assays. Analysis of SF enzymes in 19 SF samples from human and swine revealed moderate activity of e-NPPs, high activity of e-NTPDases, and low activity of TNAP. Our newly developed method for analysis of multiple enzymatic activities using Kyoto Green in biological SF will assist improvement in accuracy of the CPPD prognosis/diagnosis, which will minimize unnecessary medical procedures. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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13 pages, 2768 KiB

Open AccessFeature PaperArticle

Effect of Potassium Concentration on Triplex Stability under Molecular Crowding Conditions

by Ye Teng, Hisae Tateishi-Karimata, Tatsuya Ohyama and Naoki Sugimoto

Molecules 2020, 25(2), 387; https://doi.org/10.3390/molecules25020387 - 17 Jan 2020

Cited by 8 | Viewed by 3584

Abstract

The properties of non-canonical DNA structures, like G-quadruplexes and triplexes, change under cell-mimicking molecular crowding conditions relative to dilute aqueous solutions. The analysis of environmental effects on their stability is crucial since they play important roles in gene expression and regulation. In this [...] Read more.

The properties of non-canonical DNA structures, like G-quadruplexes and triplexes, change under cell-mimicking molecular crowding conditions relative to dilute aqueous solutions. The analysis of environmental effects on their stability is crucial since they play important roles in gene expression and regulation. In this study, three intramolecular and intermolecular triplex-forming sequences of different C⁺_*G-C triplet content (_*: Hoogsteen base pair; - : Watson–Crick base pair) were designed and their stability measured in the absence and presence of a crowding agent with different K⁺ concentrations. In dilute solution, the stability of the triplexes was reduced by decreasing the concentration of KCl. This reduction became smaller as the number of C⁺_*G-C triplets increased. Under molecular crowding conditions, Watson–Crick base pairs and Hoogsteen base pairs were destabilized and stabilized, respectively. Interestingly, with lower KCl concentrations (≤1 M), the destabilization of the triplexes due to reduction of KCl concentration was significantly smaller than in dilute solutions. In addition, the C⁺_*G-C content had greater influence on triplex stability under molecular crowding conditions. Our work provides quantitative information about the effects of K⁺ concentration on triplex stability under molecular crowding conditions and should further our understanding of the function and regulation of triplexes in bioprocesses. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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12 pages, 2012 KiB

Open AccessArticle

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death

by Katsuhiko Itoh, Takahiro Ebata, Hiroaki Hirata, Takeru Torii, Wataru Sugimoto, Keigo Onodera, Wataru Nakajima, Ikuno Uehara, Daisuke Okuzaki, Shota Yamauchi, Yemima Budirahardja, Takahito Nishikata, Nobuyuki Tanaka and Keiko Kawauchi

Molecules 2019, 24(17), 3175; https://doi.org/10.3390/molecules24173175 - 01 Sep 2019

Cited by 7 | Viewed by 4770

Abstract

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction [...] Read more.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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11 pages, 3450 KiB

Open AccessArticle

Next Generation Sequencing-Based Transcriptome Predicts Bevacizumab Efficacy in Combination with Temozolomide in Glioblastoma

by Alimu Adilijiang, Masaki Hirano, Yusuke Okuno, Kosuke Aoki, Fumiharu Ohka, Sachi Maeda, Kuniaki Tanahashi, Kazuya Motomura, Hiroyuki Shimizu, Junya Yamaguchi, Toshihiko Wakabayashi and Atsushi Natsume

Molecules 2019, 24(17), 3046; https://doi.org/10.3390/molecules24173046 - 22 Aug 2019

Cited by 6 | Viewed by 6376

Abstract

Glioblastoma (GBM), the most common and malignant brain tumor, is classified according to its isocitrate dehydrogenase (IDH) mutation status in the 2016 World Health Organization (WHO) brain tumor classification scheme. The standard treatment for GBM is maximal resection, radiotherapy, and Temozolomide (TMZ). Recently, [...] Read more.

Glioblastoma (GBM), the most common and malignant brain tumor, is classified according to its isocitrate dehydrogenase (IDH) mutation status in the 2016 World Health Organization (WHO) brain tumor classification scheme. The standard treatment for GBM is maximal resection, radiotherapy, and Temozolomide (TMZ). Recently, Bevacizumab (Bev) has been added to basic therapy for newly diagnosed GBM, and monotherapy for recurrent GBM. However, the effect of IDH1 mutation on the combination of Bev and TMZ is unknown. In this study, we performed transcriptomic analysis by RNA sequencing with next generation sequencing (NGS), a newly developed powerful method that enables the quantification of the expression level of genome-wide genes. Extracellular matrix and immune cell migration genes were mainly upregulated whereas cell cycle genes were downregulated in IDH1-mutant U87 cells but not in IDH1-wildtype U87 cells after adding Bev to TMZ. In vitro and in vivo studies were conducted for further investigations to verify these results, and the addition of Bev to TMZ showed a significant antitumor effect only in the IDH1-mutant GBM xenograft model. Further studies of gene expression profiling in IDH1 mutation gliomas using NGS will provide more genetic information and will lead to new treatments for this refractory disease. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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26 pages, 4004 KiB

Open AccessArticle

Inhibition of CpLIP2 Lipase Hydrolytic Activity by Four Flavonols (Galangin, Kaempferol, Quercetin, Myricetin) Compared to Orlistat and Their Binding Mechanisms Studied by Quenching of Fluorescence

by Ruba Nasri, Luc P R Bidel, Nathalie Rugani, Véronique Perrier, Frédéric Carrière, Eric Dubreucq and Christian Jay-Allemand

Molecules 2019, 24(16), 2888; https://doi.org/10.3390/molecules24162888 - 08 Aug 2019

Cited by 23 | Viewed by 5328

Abstract

The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition [...] Read more.

The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition effects of orlistat and four flavonols (galangin, kaempferol, quercetin and myricetin) characterized by an increasing degree of hydroxylation in B-ring, were investigated using ethyl oleate hydrolysis as the model reaction. Orlistat and kaempferol (14 µM) strongly inhibited CpLIP2 catalytic activity within 1 min of pre-incubation, by 90% and 80%, respectively. The relative potency of flavonols as inhibitors was: kaempferol > quercetin > myricetin > galangin. The results suggested that orlistat bound to the catalytic site while kaempferol interacted with W294 on the protein lid. A static mechanism of interactions between flavonols and CpLIP2 lipase was confirmed by fluorescence quenching analyses, indicating that the interactions were mainly driven by hydrophobic bonds and electrostatic forces. From the Lehrer equation, fractions of tryptophan accessibility to the quencher were evaluated, and a relationship with the calculated number of binding sites was suggested. Full article

(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)

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