Biology subjects

Hwang, J. S.

Publications and source records attributed to Hwang, J. S..

3 recordsLinked to original sources

bioRxiv2022

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at [~]18 and [~]22 g/ml of rHALT-1 purified with phosphate and acetate buffers, respectively. O_LIHALT-1 is a soluble -pore-forming toxin of 18.38 kDa. C_LIO_LIrHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography. C_LIO_LIThe cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/503828v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1b01c1borg.highwire.dtl.DTLVardef@1055147org.highwire.dtl.DTLVardef@1b2eb1org.highwire.dtl.DTLVardef@966fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@82c23forg.highwire.dtl.DTLVardef@9ede5dorg.highwire.dtl.DTLVardef@18e25aorg.highwire.dtl.DTLVardef@1b4b60dorg.highwire.dtl.DTLVardef@11c1eb7_HPS_FORMAT_FIGEXP M_TBL C_TBL

biochemistry↗

bioRxiv2022

Is proteolytic cleavage essential for the activation of Hydra pore-forming toxin, HALT-4?

The mature form of Hydra actinoporin-like toxin 4 (mHALT-4) differs from other actinoporins primarily by bearing approximately 115 additional residues at the N-terminus. Five dibasic residues were found in this extended region and we assume that, when cleaved, each truncated HALT-4 (tKK1, tKK2, tRK3, tKK4 and tKK5) could exhibit an enhanced cytolytic activity. However, our results showed that mHALT-4, tKK1 and tKK2 possessed similar cytolytic activity against HeLa cells, whereas tRK3, tKK4 and tKK5 failed to kill HeLa cells. Therefore, the cleavage of KK1 or KK2 sites did not enhance the cytolytic activity of tKK1 and tKK2 but might facilitate the sorting of tKK1 and tKK2 to the regulated secretory pathway and eventually deposit them in the nematocyst. In contrast, RK3, KK4 and KK5 were unlikely to serve as the proteolytic cleavage sites since the amino acids between KK2 and RK3 are also crucial for the pore formation. HighlightsO_LIFive dibasic cleavage sites are identified at the N-terminal region of HALT-4 but they are unlikely to have a role in the enhancement of HALT-4 cytotoxicity. C_LIO_LIThe first two dibasic sites, KK1 and KK2, may be involved in sorting HALT-4 to the regulated secretory pathway where HALT-4 can be transported to the nematocyst. C_LI

molecular biology↗

bioRxiv2021

Hierarchical Regulation of Autophagy During Adipocyte Differentiation

We previously showed that some adipogenic transcription factors such as CEBPB and PPARG directly and indirectly regulate autophagy gene expression in adipogenesis. The order and the effect of these events are undetermined. In this study, we modeled the gene expression, DNA-binding of transcriptional regulators, and histone modifications during adipocyte differentiation and evaluated the effect of the regulators on gene expression in terms of direction and magnitude. Then, we identified the overlap of the transcription factors and co-factors binding sites and targets. Finally, we built a chromatin states model based on the histone marks and studied their relation with the factors binding. Adipogenic factors differentially regulated autophagy genes as part of the differentiation program. Co-regulators associated with specific transcription factors and preceded them to the regulatory regions. Transcription factors differed in the binding time and location, and their effect on expression was either localized or long-lasting. Adipogenic factors disproportionately targeted genes coding for autophagy-specific transcription factors. To sum, a hierarchical arrangement between adipogenic transcription factors and co-factors drives the regulation of autophagy during adipocyte differentiation.

systems biology↗