Chan Gil Kim

Chrysanthemum morifolium Ramat (CM) extract protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity

ETHNOPHARMACOLOGICAL RELEVANCE Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications. AIM OF THE STUDY To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP(+)), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms. MATERIALS AND METHODS The effects of CM against MPP(+)-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells. RESULTS CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis. CONCLUSION These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinsons disease.

Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue‐ or stage‐specific activation toward certain genes such as globin in red blood cells and interleukin‐4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2‐binding peptides: HXPR, PHL, ASR and PXHXH. Protein‐database searching revealed that RE‐1 silencing factor (REST), Yin‐Yang1 (YY1) and five other proteins have one or two of these CP2‐binding motifs. Glutathione S‐transferase pull‐down and coimmunoprecipitation assays showed that two HXPR motif‐containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2s transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR‐containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2‐binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.

Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

Identification of p115 as a PLCγ1-binding protein and the role of Src homology domains of PLCγ1 in the vesicular transport☆

Abstract In order to gain further insight into the function(s) of PLCγ1, we tried to identify the binding partners that can interact with the SH223 domains of PLCγ1. Immunoscreening was performed with the purified antisera that are specific to SH223-binding proteins. Several immunoreactive clones were identified as the putative binding proteins and one of them was identified as p115. p115 was reported to be required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. The interaction between PLCγ1 and p115 was specific to carboxyl-terminal SH2 domain and SH3 domain of PLCγ1, and also confirmed by biochemical approaches such as co-immunoprecipitation, pull-down assay, and glycerol gradient fractionation. To further characterize the role of SH domains of PLCγ1 in the vesicle transport pathway, secreted form of alkaline phosphatase (SEAP) reporter assay was carried out. When the SH2 and/or SH3 domains of PLCγ1 were deleted, the secretion of SEAP was significantly reduced. These findings indicate that the SH2 and SH3 domains of PLCγ1 may play a role(s) in the process of the vesicle transport via interaction with other vesicle-associated proteins such as p115.

Cflarb Complemented the Function of Cflara to Allow Cflara Knock out Zebrafish To Normal Development

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κB activation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulated cFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of the more potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs) and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and whole-mount in situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafish using transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10-bp deletion in the first DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR. Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafish did not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analogues of both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than those in the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate compared with wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. This data suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.

Synapsin IIb interacts with the C-terminal SH2 and SH3 domains of PLCγ1 and inhibits its enzymatic activity

To elucidate the function of PLCγ1, we have investigated the proteins that bind to its SH (Src homology) domain. Immunoscreening was performed with purified antisera specific for SH223 (two SH2 and one SH3)‐binding proteins. Several immunoreactive clones were identified as putative binding proteins and one of them was identified as synapsin IIb. We demonstrate a stable association between PLCγ1 and synapsin IIb, which binds the carboxyl terminal SH2 and SH3 domains of the enzyme and inhibits it.