Stabilization by Fusion to the C-terminus of Hyperthermophile Sulfolobus tokodaii RNase HI: A Possibility of Protein Stabilization Tag

Takano, Kazufumi; Okamoto, Takahiro; Okada, Jun; Tanaka, Shun‐ichi; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

doi:10.1371/journal.pone.0016226

Cited by 16 publications

(7 citation statements)

References 33 publications

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“…The most similar structure was that of RNase HI from Sulfolobus tokodaii (Protein Data Bank (PDB) code: 3ALY, Z -score = 8.2, r.m.s.d. = 3.6 Å, sequence identity = 15%) ( 40 ). In addition, the structure of RNase HI from Bacillus halodurans in complex with an RNA/DNA hybrid (PDB code: 1ZBI, Z -score = 4.7, r.m.s.d.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I

et al. 2015

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Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3′-5′ exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3′-OH recognition site ∼9 Å away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required.

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Section: Resultsmentioning

confidence: 99%

Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I

et al. 2015

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“…Comparison of the crystal structure of LC11‐RNase H1 with that of Sto‐RNase H1ΔC620 indicates that the number of buried nonpolar residues and the number of ion pairs of LC11‐RNase H1 are comparable to those of Sto‐RNase H1ΔC6, while the number of buried charged residues and the total volume of cavities of LC11‐RNase H1 are higher than those of Sto‐RNase H1ΔC6 (Table I). These results suggest that LC11‐RNase H1 is destabilized as compared to Sto‐RNase H1 not only by removal of the C‐terminal anchoring tail but also by increasing the cavity volume and the number of buried charged residues.…”

Section: Resultsmentioning

confidence: 99%

Activity, stability, and structure of metagenome‐derived LC11‐RNase H1, a homolog of Sulfolobus tokodaii RNase H1

et al. 2012

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Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA). To examine whether LC11-RNase H1 also exhibits both RNase H and dsRNase activities, LC11-RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11-RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto-RNase H1. However, LC11-RNase H1 did not exhibit dsRNase activity at any condition examined. LC11-RNase H1 was less stable than Sto-RNases H1 and its derivative lacking the C-terminal tail (Sto-RNase H1DC6) by 37 and 13°C in T m , respectively. To understand the structural bases for these differences, the crystal structure of LC11-RNase H1 was determined at 1.4 Å resolution. The LC11-RNase H1 structure is highly similar to the Sto-RNase H1 structure. However, LC11-RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA-phosphate binding pocket, while Sto-RNase H1 has one groove containing the active site. In addition, LC11-RNase H1 contains more cavities and buried charged residues than Sto-RNase H1. We propose that LC11-RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11-RNase H1 is less stable than Sto-RNase H1DC6 because of the increase in cavity volume and number of buried charged residues.

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“…A study by Takano et al demonstrated that the fusion of C-terminal residues of Sto-RNase HI to other proteins resulted in more stable chimeric proteins. [24] For in vivo application, it was demonstrated that biomaterials could be deliberately chosen for GF-based wound regeneration based on GF structural considerations, such as ionic properties, surface charges, and binding sites. [25] We chose the carbomer under the assumption that it plays a role not only in retaining the proteins at wound sites but also in providing an electrostatic binding moiety to positively charged bFGF due to its intrinsic anionic nature.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin‐Penetrating Property of bFGF Using 30Kc19α‐Based Fusion Protein

Lee

Kim

et al. 2021

Advanced Biology

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involvement in tissue growth and regeneration, including wound healing. Activation of FGF receptor by bFGF stimulates intracellular signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways which are related to the mitogenic response of cells. [1] As bFGF acts on various target cells involved in wound healing, its effects on wound healing have been extensively studied. [2] bFGF promotes the migration of dermal fibroblasts, keratinocytes, and endothelial cells, into wound sites and facilitates their proliferation. [3] As a result, bFGF accelerates granulation tissue formation and angiogenesis, which results in matrix formation and remodeling. [4] In recent years, a number of clinical uses of recombinant bFGF have been reported, and these include treatment of chronic wounds, second-degree burns, pressure ulcers, and diabetic foot ulcers. [5] However, its inherent instability and rapid degradation often require frequent treatments in tandem with higher concentrations. [6] The reported half-life of recombinant bFGF is less than 10 h under normal cell culture conditions and even shorter in the living organisms, which significantly limits The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it is investigated whether fusing 30Kc19α to bFGF can enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increases protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retains the biological activity of bFGF as it facilitates the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It is discovered that 30Kc19α can improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increases the accumulation of bFGF and further facilitates its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrates the dermis layer effectively, which promotes cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, the findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

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Stabilization by Fusion to the C-terminus of Hyperthermophile Sulfolobus tokodaii RNase HI: A Possibility of Protein Stabilization Tag

Cited by 16 publications

References 33 publications

Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I

Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I

Activity, stability, and structure of metagenome‐derived LC11‐RNase H1, a homolog of Sulfolobus tokodaii RNase H1

Enhancement of Wound Healing Efficacy by Increasing the Stability and Skin‐Penetrating Property of bFGF Using 30Kc19α‐Based Fusion Protein

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