Hyun-Ju Lee

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor.

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD+–cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD+ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD+ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD+, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD+ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.

The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

Crystal structure of the transcriptional activator HlyU from Vibrio vulnificus CMCP6.

MINT‐7710072, MINT‐7710086: HlyU_Vv (uniprotkb:Q8DES3) and HlyU_Vv (uniprotkb:Q8DES3) bind (MI:0407) by X‐ray crystallography (MI:0114)

Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in a Model of Murine Multiple Myeloma.

We investigated the efficacy of lenalidomide (LEN) in combination with dendritic cell (DC) vaccination in the MOPC-315 murine myeloma model. After tumor growth, LEN was injected intraperitoneally for 4 consecutive days in combination with DC vaccination. The combination of LEN and vaccination efficiently inhibited tumor growth compared with the single agents alone. A cytotoxic assay revealed that the anticancer effects of DC vaccination plus LEN involved not only generation of antigen-specific cytotoxic T lymphocytes but also NK cells. Vaccinated mice had reduced numbers of suppressor cells, including both myeloid-derived suppressor cells and regulatory T cells, in the spleen. The proportions of CD4+ and CD8+ T cells increased in the spleen, and a Th1 cytokine (interferon-&ggr;) rather than a Th2 cytokine (interleukin-10) was synthesized in response to tumor antigens. LEN enhanced the innate immune response by modulating NK cell numbers and function. In addition, LEN reduced the production levels of angiogenesis-inducing factors in tumor-bearing mice. Together, these results suggest that a combination of LEN and DC vaccination may synergistically enhance anticancer immunity in the murine myeloma model, by inhibiting immunosuppressor cells and stimulating effector cells, as well as effectively polarizing the Th1/Th2 balance in favor of a Th1-specific immune response.

Dendritic cells loaded with myeloma cells pretreated with a combination of JSI-124 and bortezomib generate potent myeloma-specific cytotoxic T lymphocytes in vitro

Signal transducer and activator of transcription 3 (STAT3) is highly activated in multiple myeloma. Activated STAT3 promotes survival and proliferation of cancer cells, suppresses Th1 immune response, and induces dysfunction of immune cells. We investigated whether pretreating myeloma cells with a phosphor (p)-STAT3 inhibitor (JSI-124) and/or bortezomib before loading into dendritic cells (DCs) can affect DC function. The combination treatment with JSI-124 and bortezomib resulted in the highest expression of heat shock protein (HSP) 90 and the lowest expression of p-STAT3 in dying myeloma cells. DCs loaded with dying myeloma cells treated by JSI-124 and bortezomib produced the least amount of p-STAT3 compared to other treatments. The DCs were recovered from abnormal cytokine secretions of interleukin (IL)-10, IL-6, and IL-23 without any effect on production of IL-12p70. DCs loaded with JSI-124 and bortezomib treated, dying myeloma cells most potently generated myeloma-specific cytotoxic T lymphocytes (CTLs). The data suggest that pretreatment of myeloma cells with JSI-124 and bortezomib can recover DC function through the up-regulation of HSP90 and the down-regulation of p-STAT3 and inhibitory cytokines, and that these DCs can potently generate myeloma-specific CTLs.

A bacterial flagellin in combination with proinflammatory cytokines activates human monocyte-derived dendritic cells to generate cytotoxic T lymphocytes having increased homing signals to cancer.

Flagellin, the cognate ligand for toll-like receptor 5, has potent adjuvant activity in various vaccines. However, its efficacy in generating dendritic cells (DCs) remains contentious. This study assessed how efficaciously Vibrio vulnificus FlaB (v-FlaB) could be used in generating a potent DC to induce antigen-specific cytotoxic T lymphocytes (CTLs). Mature DCs (mDCs) induced by the combination of v-FlaB/TNF&agr;/IFN&agr; were significantly more potent in inducing specific anticancer immune responses compared with the standard DCs that were maturated by the conventional cytokine cocktail of TNF&agr;/IL-1&bgr;/IL-6/PGE2. The potent mDCs produced a higher level of interleukin (IL)-12p70 and polarized naive CD4+ T cells more towards Th1-type cells, markedly increased antigen-specific CD8+ T-cell number and significantly enhanced the induction of lytic enzymes in antigen-specific CD8+ CTLs and sensitized CD3+ T cells to produce higher number of interferon (IFN)&ggr;-secreting cells. As a result, the mDCs produced more potent antigen-specific CTLs against the MART-1 and expressed higher levels of homing receptors CCR5 and CXCR3. More importantly, the v-FlaB/TNF&agr;/IFN&agr;-DCs generated from melanoma patients produced strong autologous CTLs with efficient cytotoxic activities. In conclusion, v-FlaB combined with tumor necrosis factor (TNF)&agr; and IFN&agr; can generate potent DCs which produce functionally active CTLs and that may have potential as a potent cancer vaccine.

Generation of Potent Cytotoxic T Lymphocytes Against Castration‐Resistant Prostate Cancer Cells by Dendritic Cells Loaded With Dying Allogeneic Prostate Cancer Cells

To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)‐based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration‐resistant prostate cancer (CRPC) using prostate cancer–specific CTLs generated in vitro by DCs. Monocyte‐derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL‐1β, TNF‐α, IL‐6 and PGE2: standard DCs, sDCs) or using an α‐type 1‐polarized DC (αDC1) cocktail (in IL‐1β, TNF‐α, IFN‐α, IFN‐γ and polyinosinic:polycytidylic acid) and loaded with the UVB‐irradiated CRPC cell line PC‐3. Antigen‐loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin‐12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer–specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC‐3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC‐specific CTL responses as compared to sDCs and may provide a novel source of DC‐based vaccines that can be used for the development of immunotherapy in patients with CRPC.

Generation of Multiple Peptide Cocktail-Pulsed Dendritic Cells as a Cancer Vaccine

Cancer immunotherapy based on dendritic cell (DC) vaccination has promising alternatives for the treatment of cancer. A central tenet of DC-based cancer immunotherapy is the generation of antigen-specific cytotoxic T lymphocyte (CTL) response. Tumor-associated antigens (TAA) and DC play pivotal roles in this process. DCs are well known to be the most potent antigen-presenting cells and have the most powerful antigen-presenting capacity. DCs pulsed with various TAA have been shown to be effective in producing specific antitumor effects both in vitro and in vivo. Several types of tumor antigens have been applied in cancer treatment including tumor RNA, lysates, apoptotic bodies, heat shock protein, peptides from TAA, and allogeneic tumor cells. Among them, the use of immunogenic HLA-A*0201-specific epitopes from multiple TAA enhances induction of antigen-specific CTL and associated therapeutic efficacy in HLA-A*0201(+) cancer patients. The current chapter provides a detailed protocol of generating multiple peptide cocktail-pulsed DC to elicit CTL with a broad spectrum of immune responses against the related tumor antigens.

Structural characterization of a modification subunit of a putative type I restriction enzyme from Vibrio vulnificus YJ016.

In multifunctional type I restriction enzymes, active methyltransferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit from Vibrio vulnificus YJ016 (vvHsdM) was elucidated at a resolution of 1.80 Å. A cofactor-binding site for S-adenosyl-L-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an α/β-fold, in which a number of residues are conserved, including the GxGG and (N/D)PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase of Thermus aquaticus and other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group.

Gesture recognition in video image with combination of partial and global information

In this paper, we describe an algorithm which can automatically recognize human gesture in a sequence of natural video image by utilizing two dimensional features extracted from bodily region of the images. In the algorithm, we first construct a gesture model space by analyzing the statistical information of sample images with principle component analysis method. And then, input images are compared to the model and individually symbolized to one part of the model space. In the last step, the symbolized images are recognized with HMM as one of model gestures. The feature of our method is to use a combination of partial and global information of two-dimensional abstract bodily motion, consequently it is very convenient to apply to real world situation and the recognition results are very robust.