Browsing by Author "Imasaki, Tsuyoshi"

Now showing 1 - 6 of 6
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    Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells
    (Elsevier, 2013) Khanna, May; Imasaki, Tsuyoshi; Chikwana, Vimbai M.; Perez-Miller, Samantha; Hunter, Gerald O.; Mosley, Amber; Takagi, Yuichiro; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of Medicine
    We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
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    High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity
    (Springer Nature, 2019-09-17) Zhu, Wen; Radadiya, Ashish; Bisson, Claudine; Wenzel, Sabine; Nordin, Brian E.; Martínez-Márquez, Francisco; Imasaki, Tsuyoshi; Sedelnikova, Svetlana E.; Coricello, Adriana; Baumann, Patrick; Berry, Alexandria H.; Nomanbhoy, Tyzoon K.; Kozarich, John W.; Jin, Yi; Rice, David W.; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
    Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 μM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.
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    MultiBac: expanding the research toolbox for multiprotein complexes
    (Elsevier, 2012-02) Bieniossek, Christoph; Imasaki, Tsuyoshi; Takagi, Yuichiro; Berger, Imre; Biochemistry and Molecular Biology, School of Medicine
    Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.
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    A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells
    (Wiley, 2019) Wenzel, Sabine; Imasaki, Tsuyoshi; Takagi, Yuichiro; Biochemistry and Molecular Biology, School of Medicine
    The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression.
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    Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system
    (Public Library of Science, 2018-04-03) Imasaki, Tsuyoshi; Wenzel, Sabine; Yamada, Kentaro; Bryant, Megan L.; Takagi, Yuichiro; Biochemistry and Molecular Biology, School of Medicine
    The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.
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    Yeast Hrq1 shares structural and functional homology with the disease-linked human RecQ4 helicase
    (Oxford University Press, 2017-05-19) Rogers, Cody M.; Wang, Joseph Che-Yen; Noguchi, Hiroki; Imasaki, Tsuyoshi; Takagi, Yuichiro; Bochman, Matthew L.; Biochemistry and Molecular Biology, School of Medicine
    The five human RecQ helicases participate in multiple processes required to maintain genome integrity. Of these, the disease-linked RecQ4 is the least studied because it poses many technical challenges. We previously demonstrated that the yeast Hrq1 helicase displays similar functions to RecQ4 in vivo, and here, we report the biochemical and structural characterization of these enzymes. In vitro, Hrq1 and RecQ4 are DNA-stimulated ATPases and robust helicases. Further, these activities were sensitive to DNA sequence and structure, with the helicases preferentially unwinding D-loops. Consistent with their roles at telomeres, telomeric repeat sequence DNA also stimulated binding and unwinding by these enzymes. Finally, electron microscopy revealed that Hrq1 and RecQ4 share similar structural features. These results solidify Hrq1 as a true RecQ4 homolog and position it as the premier model to determine how RecQ4 mutations lead to genomic instability and disease.