Bovine Serum Albumin Hydrogel Formation: pH Dependence and Rheological Analyses.

In vitro evaluation of the physical properties of biopolymer-based hydrogels can help in understanding certain phenomena, such as liquid-liquid phase separation. The formation of bovine serum albumin (BSA) hydrogels was investigated in the pH range of 1.0 to 4.0. Hydrogels were formed in the pH range of 3.0 to 4.0, whereas viscous solutions were formed in the pH range of 1.5 to 2.5. Unexpectedly, formation of BSA hydrogel was observed in extremely acidic condition (pH 1.0). The circular dichroism spectra of BSA solutions were recorded at pH values of 1.0, 2.0, 3.0, and 7.0, and α-helix contents were determined from the ellipticity data at 222 nm. The α-helix content decreased with a decrease in pH, and this decrease was associated with the partial denaturation (F-isoform) and the denaturation (E-isoform) of BSA. However, the α-helix contents at pH 1.0 and 3.0 were similar. BSA hydrogels at pH 1.0 and 3.5 showed similar dynamic viscoelastic properties, further supporting the stereo structural change of BSA from the denatured E-isoform to the partially denatured F-isoform at pH 1.0. The study also focused on measuring viscoelasticity, a fundamental physical property of hydrogels, using traditional rheometer and with minimal sample volume. A highly reproducible procedure for measuring the viscoelastic properties of hydrogels was established using sample volumes of 200 and 350 µL.

DNA G-Quadruplex-Binding Protein Developed Using the RGG Domain of Translocated in Liposarcoma/Fused in Sarcoma Inhibits Transcription of bcl-2.

The G-quadruplexes (G4s) in the genome are important drug targets because they regulate gene expression and the genome structure. Several small molecules that bind the G4 have been developed, but few artificial G4 binding proteins have been reported. We previously reported a novel DNA G4 binding protein (RGGF) engineered using the Arg-Gly-Gly repeat (RGG) domain of TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma) protein (TLS/FUS). Here, we show that RGGF recognizes DNA loops in the G4 and preferentially binds DNA G4 with long loops in vitro. Furthermore, RGGF binds to the DNA G4 of the bcl-2 promoter in vitro. RGGF overexpression in HeLa cells represses bcl-2 transcription. On the basis of these findings, G4 binding protein engineered from the RGG domain will be useful for investigating G4 transcriptional function in the genome.

Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14.

N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5'-DRACH-3' in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.

G-quadruplex-proximity protein labeling based on peroxidase activity.

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.

Oligonucleotides DNA containing 8-trifluoromethyl-2'-deoxyguanosine for observing Z-DNA structure.

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2'-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA.

Roles of the RGG Domain and RNA Recognition Motif of Nucleolin in G-Quadruplex Stabilization.

G-quadruplexes have important biologic functions that are regulated by G-quadruplex-binding proteins. In particular, G-quadruplex structures are folded or unfolded by their binding proteins and affect transcription and other biologic functions. Here, we investigated the effect of the RNA recognition motif (RRM) and arginine-glycine-glycine repeat (RGG) domain of nucleolin on G-quadruplex formation. Our findings indicate that Phe in the RGG domain of nucleolin is responsible for G-quadruplex binding and folding. Moreover, the RRM of nucleolin potentially binds to a guanine-rich single strand and folds the G-quadruplex with a 5'-terminal and 3'-terminal single strand containing guanine. Our findings contribute to our understanding of how the RRM and RGG domains contribute to G-quadruplex folding and unfolding.

Modulation of histone modifications and G-quadruplex structures by G-quadruplex-binding proteins.

The functions of local conformations of non-B form DNA and RNA, such as the G-quadruplex, are thought to be regulated by their specific binding proteins. They regulate the formation of G-quadruplexes in cells and affect the biological functions of G-quadruplexes. Recent studies reported that G-quadruplexes regulate epigenetics through these G-quadruplex binding proteins. We discuss regulation of histone modifications through G-quadruplex RNA and its binding proteins which modulate the G-quadruplex conformations. G-quadruplex RNA is involved in telomere maintenance and transcription via histone modification. Furthermore, G-quadruplex binding proteins regulate formation and biological functions of G-quadruplexes through regulating their folding or unfolding. In this review, we will focus on the G-quadruplex binding proteins containing RRM and RGG domains.

RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS.

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.

Stereoselective synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres and their application to 14-mer RGG peptidomimetics.

Stereoselective and efficient synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres is realized by organocuprate-mediated single electron transfer reduction. The synthetic isosteres can be used in Fmoc-based solid phase peptide synthesis, resulting in the preparation of the 14-mer RGG peptidomimetics containing an (E)-methylalkene or a (Z)-chloroalkene unit.

Heterologous production of coryneazolicin in Escherichia coli.

Coryneazolicin is a plantazolicin family peptide, belonging to linear azole-containing peptides (LAPs). Although coryneazolicin was previously synthesized by in vitro experiments, its biological activity has not been evaluated. In this report, the heterologous production of coryneazolicin was accomplished to obtain enough coryneazolicin for biological activity tests. The structure of coryneazolicin was confirmed by ESI-MS and NMR analyses. The biological activity tests indicated that coryneazolicin possessed potent antibacterial activity and cytotoxicity. Although antibacterial activity of plantazolicin was previously reported, cytotoxicity was newly found in coryneazolicin among plantazolicin type peptides. In addition, we revealed that coryneazolicin induced apoptosis on HCT116 and HOS cancer cell lines.

Isolation and structure determination of a new cytotoxic peptide, curacozole, from Streptomyces curacoi based on genome mining.

Using genome mining, a new cytotoxic peptide named curacozole was isolated from Streptomyces curacoi. Through ESI-MS and NMR analyses, curacozole was determined to be a macrocyclic peptide containing two isoleucine, two thiazole and three oxazole moieties. Curacozole exhibited potent cytotoxic activity against HCT116 and HOS cancer cells. The proposed biosynthetic gene cluster of curacozole was identified and compared with that of the related compound YM-216391.

G-quadruplex binding ability of TLS/FUS depends on the ß-spiral structure of the RGG domain.

The RGG domain, defined as closely spaced Arg-Gly-Gly repeats, is a DNA and RNA-binding domain in various nucleic acid-binding proteins. Translocated in liposarcoma (TLS), which is also called FUS, is a protein with three RGG domains, RGG1, RGG2 and RGG3. TLS/FUS binding to G-quadruplex telomere DNA and telomeric repeat-containing RNA depends especially on RGG3, comprising Arg-Gly-Gly repeats with proline- and arginine-rich regions. So far, however, only non-specific DNA and RNA binding of TLS/FUS purified with buffers containing urea and KCl have been reported. Here, we demonstrate that protein purification using a buffer with high concentrations of urea and KCl decreases the G-quadruplex binding abilities of TLS/FUS and RGG3, and disrupts the ß-spiral structure of RGG3. Moreover, the Arg-Gly-Gly repeat region in RGG3 by itself cannot form a stable ß-spiral structure that binds to the G-quadruplex, because the proline- and arginine-rich regions induce the ß-spiral structure and the G-quadruplex-binding ability of RGG3. Our findings suggest that the G-quadruplex-specific binding abilities of TLS/FUS require RGG3 with a ß-spiral structure stabilized by adjacent proline- and arginine-regions.

Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA.

The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

Bcl-XL-binding helical peptides possessing d-Ala residues at their C-termini with the advantage of long-lasting intracellular stabilities.

We attached d-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The d-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-XL. The d-Ala attached helical peptides were much more stable in cells than original ones and efficiently induced apoptosis of the cells.

Supramolecular gel electrophoresis of large DNA fragments.

Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system.

Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, ß, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, ß, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, ß, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

G-Quadruplex DNA- and RNA-Specific-Binding Proteins Engineered from the RGG Domain of TLS/FUS.

Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.

Calculation of the HOMO localization of Tetrahymena and Oxytricha telomeric quadruplex DNA.

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3'-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5'-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K(+) and d(TAGGGT)4T +2K(+) localized at the 5'-guanine, and that the HOMO shifted from the 5'-guanine to the 3'-guanine by the addition of a 5'-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K(+) and d(TAGGGT)4T +2K(+) matched the experimental results and suggest that d(TGGGGT)4 contains a 5'-capping cation in solution.

Tailor-made designer helical peptides that induce mitochondrion-mediated cell death without necrosis.

Managing protein-protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side-chain-crosslinked helical peptides based on the binding domain of a pro-apoptotic protein (Bad) that induces programmed cell death. The peptide showed high helical content and bound to its target, Bcl-XL, more strongly than its non-crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl-XL localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70% of HeLa cells died by the apoptosis pathway (without evidence of necrosis).

Supramolecular gel electrophoresis of acidic native proteins.

Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane-glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material.