Sayeon Cho

NMR-based metabolic profiling and differentiation of ginseng roots according to cultivation ages

Ginseng is an important herbal resource worldwide, and the adulteration or falsification of cultivation age has been a serious problem in the commercial market. In this study, ginseng (Panax ginseng) roots, which were cultivated for 2-6 years under GAP standard guidelines, were analyzed by NMR-based metabolomic techniques using two solvents. At first, ginseng root samples were extracted with 50% methanol, and analyzed by NMR with D(2)O as the NMR dissolution solvent. The 2-, 3-, 4-, and 5/6-year-old ginseng root samples were separated in PLS-DA-derived score plots. However, 5- and 6-year-old ginseng roots were not separated by the solvent system. Therefore, various solvents were tested to differentiate the 5- and 6-year-old ginseng root samples, and 100% methanol-d(4) was chosen as the direct extraction and NMR dissolution solvent. In the PLS model using data from the 100% methanol-d(4) solvent, 5- and 6-year-old ginseng roots were clearly separated, and the model was validated using internal and external data sets. The obtained RMSEE and RMSEP values suggested that the PLS model has strong predictability for discriminating the age of 5- and 6-years-old ginseng roots. The present study suggests that the age of ginseng could be successfully predicted using two solvents, and the developed method in this study can be used as a standard protocol for discriminating and predicting the ages of ginseng root samples.

Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction

Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin‐related protein, functions to facilitate the hydrolysis of 5‐hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin‐related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.

Binding and regulation of HIF-1α by a subunit of the proteasome complex, PSMA7

The hypoxia‐inducible factor‐1α (HIF‐1α) is an important transcription factor for cellular responses to oxygen tension. It is rapidly degraded under normoxic conditions by the ubiquitin‐dependent proteasome pathway. Here we report a critical role of the 20S proteasome subunit PSMA7 in HIF‐1α regulation. PSMA7 was found to interact specifically with two subdomains of HIF‐1α. PSMA7 inhibited the transactivation function of HIF‐1α under both normoxic and hypoxia‐mimicking conditions. In addition, we show that the PSMA7‐mediated regulation of HIF‐1α activity is associated with the proteasome pathway.

Annexin A4 interacts with the NF-κB p50 subunit and modulates NF-κB transcriptional activity in a Ca2+-dependent manner

Previously, we identified annexin A4 (ANXA4) as a candidate substrate of caspase-3. Proteomic studies were performed to identify interacting proteins with a view to determining the roles of ANXA4. ANXA4 was found to interact with the p105. Subsequent studies revealed that ANXA4 interacts with NF-κB through the Rel homology domain of p50. Furthermore, the interaction is markedly increased by elevated Ca2+ levels. NF-κB transcriptional activity assays demonstrated that ANXA4 suppresses NF-κB transcriptional activity in the resting state. Following treatment with TNF-α or PMA, ANXA4 also suppressed NF-κB transcriptional activity, which was upregulated significantly early after etoposide treatment. This difference may be due to the intracellular Ca2+ level. Additionally, ANXA4 translocates to the nucleus together with p50, and imparts greater resistance to apoptotic stimulation by etoposide. Our results collectively indicate that ANXA4 differentially modulates the NF-κB signaling pathway, depending on its interactions with p50 and the intracellular Ca2+ ion level.

The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1α

Hepatitis B virus X protein (HBx) of the hepatitis B virus is strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Previously, we reported that HBx enhances activity of hypoxia‐inducible factor‐1α (HIF‐1α), a potent transactivator that induces angiogenic factors. Here, we delineate the structural region of HBx that potentiates HIF‐1α. The carboxy‐terminus of HBx increased the stability of HIF‐1α protein, probably through inhibiting interaction with von Hippel‐Lindau protein. Further, the carboxy‐terminus of HBx enhanced the transactivation function of HIF‐1α by enhancing its association with CREB binding protein (CBP). Finally, we demonstrated the physical association of HBx with the basic helix–loop–helix/PER–ARNT–SIM domain, the inhibitory domain, and the carboxy‐terminal transactivation domain of HIF‐1α in vivo.

Co-chaperone CHIP promotes aggregation of ataxin-1

Recent studies demonstrated that co-chaperone/E3 ligase CHIP (C-terminus of hsp70-interacting protein) mediates the ubiquitylation and suppresses the aggregation of polyglutamine (polyQ) proteins, such as huntingtin or ataxin-3. In this study, we investigated the effects of CHIP on the degradation of another polyQ protein ataxin-1. Interestingly CHIP associates not only with the polyQ-expanded ataxin-1 but also with the normal ataxin-1. Moreover, by enhancing ataxin-1 ubiquitylation, CHIP over-expression leads to a reduction in the solubility of ataxin-1 and thus increases the aggregate formation, especially that of polyQ-expanded ataxin-1. Domain analysis revealed that the TPR domain is required for the promotion of aggregation. By contrast, other co-chaperones or E3 ligases, such as BAG-1 or parkin, did not show similar effects on the aggregation of ataxin-1. Importantly, the effect of CHIP is impaired by the mutation of Ser776 of ataxin-1 whose phosphorylation is crucial for ataxin-1 aggregation. Our findings suggest that the role of CHIP in aggregation of polyQ proteins greatly varies depending on the context of full-length polyQ proteins.

Oxidative stress-enhanced SUMOylation and aggregation of ataxin-1: Implication of JNK pathway.

Although the polyglutamine protein ataxin-1 is modified by SUMO at multiple sites, the functions of such modification or how it is regulated are still unknown. Here we report that SUMO-1 or Ubc9 over-expression stimulated the aggregation of ataxin-1 and that oxidative stress, such as hydrogen peroxide treatment, further enhanced SUMO conjugation and aggregation of ataxin-1. Accordingly, co-treatment with antioxidant N-acetyl-cysteine attenuated the effect of oxidative stress. Ataxin-1, which can activate c-Jun N-terminal kinase (JNK) pathway by itself, strongly associated with apoptosis signal-regulating kinase 1 (ASK1) while not interacting with JNK. Finally, treatment of JNK-specific inhibitor caused a reduction in the oxidant-enhanced SUMOylation and aggregation of ataxin-1. Together these results indicate that SUMO modification of ataxin-1 promotes the aggregation of ataxin-1 and that oxidative stress and JNK pathway play roles in this process.

NSC-87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26)

Protein phosphorylation plays critical roles in many regulatory mechanisms controlling cell activities and thus involved in various diseases. The cellular equilibrium of phosphorylation is regulated through the actions of protein kinases and phosphatases. Therefore, these regulatory proteins have emerged as promising targets for drug development. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), a potent inhibitor of SHP-1 and SHP-2 PTPs. Phosphatase activity of dual-specificity protein phosphatase 26 (DUSP26) was decreased by the inhibitor in a dose-dependent manner. Kinetic studies with NSC-87877 and DUSP26 revealed a competitive inhibition. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 could be a therapeutic reagent for treating ATC.

A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography.

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and the other for zymography, were run in parallel. After silver staining of SDS‐PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix‐assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two‐dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.

Activation of autophagy during glutamate-induced HT22 cell death

Recent evidence suggests that autophagy plays a role in oxidative injury-induced cell death. Here we examined whether glutamate-mediated oxidative toxicity induces autophagy in murine hippocampal HT22 cells and if autophagy induction affects the molecular events associated with cell death. Markers for autophagy induction including LC3 conversion, suppression of mTOR pathway, and GFP-LC3 dot formation were enhanced by glutamate treatment. By contrast, autophagy inhibition blocked glutamate-induced LC3 conversion and consequently reduced cell death. Activation of ERK1/2, a hallmark of glutamate-induced cytotoxicity, was also decreased by autophagy inhibition. Interestingly, autophagy inhibition also affected the expression of chaperones including Hsp60 and Hsp70, which are differentially regulated during HT22 cell death. Conversely, knock-down of Hsp60 greatly decreased LC3 conversion. Together these results suggest that glutamate-induced cytotoxicity involves autophagic cell death and chaperones may play a role in this process.