Biology subjects

Kuroda, Y.

Publications and source records attributed to Kuroda, Y..

23 records · Page 2Linked to original sources

bioRxiv2020

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Gaussia luciferase (GLuc) is the smallest luciferase (18.2kDa; 168 residues) reported so far and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a solubility tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Thermodynamic analysis of point mutations inhibiting high-temperature reversible oligomer of PDZ3

Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation exhibiting two endothermic peaks due to the formation of reversible oligomers at high temperature (N{leftrightarrow}I6{leftrightarrow}D). This contrasts sharply with the standard 2-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect 2-state denaturation, as evidenced by the single endothermic peak, and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. While we designed the mutations based on our previous study showing that a point mutation Val380 to a nonhydrophobic amino acid inhibited RO in DEN4 ED3, the results are nevertheless surprising since high-temperature RO involves proteins in a denatured state, as assessed by circular dichroism. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation. SignificanceDespite being a small globular protein, which normaly undergo a two-state unfolding, the thermal denaturation of PSD95-PDZ3, monitored by DSC, exhibited two endothermic peaks. The second peak resulted from a reversible oligomerization (RO) at high temperatures, which is, on its own, a rare phenomenon. In this study, we show that the substitution of a single hydrophobic residue to an alanine at the interface of the crystallographic tetramers inhibited high-temperature RO, resulting in a single endothermic peak. Future studies are required to determine why mutations designed using crystal structures determined at ambient temperatures can inhibit high-temperature RO, and whether the ROs are precursor of irreversible aggregation, If so, the present observations will provide an entirely new basis for creating aggregation-resistant proteins.

biophysics↗

bioRxiv2020

A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein 240 folds

Small proteins are generally non-immunogenic, which can be a major hurdle in developing protein and peptide vaccines or producing antibodies for biopharmaceutical usage. For improving a proteins immunogenicity, we previously proposed to use short Solubility Controlling Peptide (SCP) tags that oligomerize proteins into soluble aggregates. Here, we systematically analyzed the effect of SCP-tags that do not induce oligomerization on the immunogenicity of a small, non-immunogenic, model protein, Bovine Pancreatic Trypsin Inhibitor (BPTI-19A; 6 kDa). We assessed the effect of the following ten SCP-tags: Six tags made of five consecutive Arg, Lys, His, Asp, Asn, Pro; one made of seven Pro; two tags made of consecutive Arg-lle and Asn-Ile, all attached at the C-terminus of BPTI-19A; and a 5-proline tag attached at the N-terminus. Circular dichroism, fluorescence, dynamic light scattering measurements, and analytical ultra-centrifugation indicated that the addition of the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. On the other hand, the C-terminus 5-proline (C5P) tag unexpectedly increased the immunogenicity (IgG level) of BPTI-19A by up to 240 fold as assessed by ELISA. Additionally, the 5-arginine tag (C5R) increased the titer by up to 73 fold. The titer increase lasted for several weeks, and the effect was cumulative to that of the Freunds adjuvant, which is commonly used to boost a proteins immunogenicity. Altogether, SCP-tags that do not oligomerize proteins substantially increased the immunogenicity of a non-immunogenic protein, suggesting that the 5-proline and the 5-arginine SCP-tags may provide a novel tool for facilitating the production of antibodies or improving the effectiveness of protein-based vaccines.

immunology↗

bioRxiv2020

Systematic mutational analysis of epitope-grafted ED3's immunogenicity reveals a DENV3-DENV4 bi-serospecific ED3 mutant

Dengue viruses are classified into four serotypes (DENV1[~]4), and the severe forms of dengue disease, the dengue hemorrhagic fever and shock syndrome, are caused by sero-cross-reacting antibodies. However, the residue determinants of the serospecificity and sero-cross-reactivity are yet to be identified. Here, we report an epitope grafting mutational analysis of the serospecificity and cross-serospecificity of the envelope protein domain 3 (ED3; 107 residues, [~]11.6kDa), which contains two major putative epitopes of DENVs. To this end, we constructed ED3 from DENV3 (3ED3) and DENV4 (4ED3), and six epitope-grafted variants, where we transferred epitope 1 (L304I, K305D, V309M, and S310A) and/or epitope 2 (D383N, K384S, K387T, and N389H) of 4ED3 onto 3ED3 and vice versa. Mice immunization using 3ED3 and 4ED3 generated serotype-specific antisera, as expected. Similarly, most epitope-grafted ED3s produced antisera serospecific to the template ED3 with little or no cross-recognition of ED3 of the serotype from which the epitopes were taken. This result indicated that a mere grafting of the epitope was not sufficient to transfer serospecificity, contrary to our expectations. However, one epitope grafted ED3 mutant, where epitope 1 of 3ED3 was grafted onto 4ED3 (4ED3epi1), generated antisera that was serospecific to both 4ED3 and 3ED3. The 4ED3epi1 is thus a chimeric ED3 that produces antisera possessing serospecificity to both 3ED3 and 4ED3. The 4ED3epi1 provides a unique tool for analyzing serospecificity and cross-reactivity in dengue, and we hope it will serve as a template for trivalent and eventually tetravalent antisera.

immunology↗

bioRxiv2019

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, resulting in continuous cell division that is a crucial factor for malignant transformation. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is a new functional unit that suppresses hTERT transcription, and plays a crucial role in cancer development through regulation of telomerase activity.

cancer biology↗