Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon

Jaturontakul, Krisadaporn; Jatuyosporn, Thapanan; Laohawutthichai, Pasunee; Kim, Sunyong; Mori, Tomoyuki; Supungul, Premruethai; Hakoshima, Toshio; Tassanakajon, Anchalee; Krusong, Kuakarun

doi:10.1038/s41598-017-06653-7

Cited by 8 publications

(7 citation statements)

References 48 publications

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“…The sedimentation velocities obtained from multiple scans at 280 nm and at different time intervals were fitted to a continuous c(s) distribution model using the SEDFIT program (36,37) to obtain the corresponding MW. The partial-specific volume of the protein sample was set as 0.73 ml/g, based on a solvent density of 1.035 g/ml (48).…”

Section: Analyses Of the Mws Oligomeric States And Quaternary Structuresmentioning

confidence: 99%

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

Ubonprasert

Jaroensuk

Pornthanakasem

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma BanerjeeHuman cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/⌬flap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/⌬flap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ␤-hydroxyamino acid. hcSHMT/⌬flap was less sensitive to THF inhibition than the WT (K i of 0.65 and 0.27 mM THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/⌬flap, indicating that the flap is important for THF binding. hcSHMT/ ⌬flap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.Serine hydroxymethyltransferase (SHMT 2 ; EC 2.1.2.1.) is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme in which the PLP cofactor facilitates the reversible transfer of a hydroxymethyl group from L-serine to (6S)-tetrahydrofolate (THF) to yield glycine and 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF) as products (1-3). SHMT is one of the enzymes in the deoxythymidylate (dTMP) synthesis cycle in which the other two enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), also take part in the recycling of folate compounds and production of 5,10-CH 2 -THF via the SHMT reaction. 5,10-CH 2 -THF serves as a methyl donor for the TS reaction to convert dUMP to dTMP, which is a requisite precursor for DNA biosynthesis (4). Due to its important role in DNA biosynthesis, cell proliferation, and cell survival, SHMT is one of the attractive targets for antimalarial (5-12) and anticancer chemotherapy (10,(13)(14)(15)(16).

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Section: Analyses Of the Mws Oligomeric States And Quaternary Structuresmentioning

confidence: 99%

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

Ubonprasert

Jaroensuk

Pornthanakasem

et al. 2019

Journal of Biological Chemistry

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“…WSSV copy number was determined as described in Jaturontakul et al . 54 . ( E ) Cumulative mortality of shrimp after challenged with WSSV.…”

Section: Resultsmentioning

confidence: 99%

“…WSSV was prepared from the gill tissue of WSSV-infected moribund shrimp using centrifugation and membrane filtration as described in Jaturontakul et al . 54 .…”

Section: Methodsmentioning

confidence: 99%

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Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon

Jatuyosporn

Laohawutthichai

Supungul

et al. 2019

Sci Rep

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White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, β2, μ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2β and μ subunits of P. monodon are reported. PmAP-2 β was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2β delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2β co-localized with WSSV particles at plasma membrane. In addition, PmAP-2β-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2β is associated with the JAK/STAT and the Toll pathway.

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“…AUC methods have been successfully applied previously for the study of integral membrane proteins and/or their supporting environments and for membrane-exporting proteins with high α-helical content (e.g. Schüler et al 2000 ; Inagaki et al 2013 ; Le Roy et al 2015 ; Surya and Torres 2015 ; Xu et al 2015 ; Lee et al 2016 ; Inagaki & Ghirlando 2017 ; Jaturontakul et al 2017 ). We showed direct interactions by vancomycin with purified intact VanS (Fig.…”

Section: Discussionmentioning

confidence: 99%

Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions

et al. 2018

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Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies.

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Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon

Cited by 8 publications

References 48 publications

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon

Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions

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