• The Korean Journal of Physiology and Pharmacology
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• 제18권6호
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• pp.517-524
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• 2014

Phasic and tonic ${\gamma}$-aminobutyric acidA ($GABA_A$) receptor-mediated inhibition critically regulate neuronal information processing. As these two inhibitory modalities have distinctive features in their receptor composition, subcellular localization of receptors, and the timing of receptor activation, it has been thought that they might exert distinct roles, if not completely separable, in the regulation of neuronal function. Inhibition should be maintained and regulated depending on changes in network activity, since maintenance of excitation-inhibition balance is essential for proper functioning of the nervous system. In the present study, we investigated how phasic and tonic inhibition are maintained and regulated by different signaling cascades. Inhibitory postsynaptic currents were measured as either electrically evoked events or spontaneous events to investigate regulation of phasic inhibition in layer 2/3 pyramidal neurons of the rat visual cortex. Tonic inhibition was assessed as changes in holding currents by the application of the $GABA_A$ receptor blocker bicuculline. Basal tone of phasic inhibition was maintained by intracellular $Ca^{2+}$ and $Ca^{2+}$/calmodulin-dependent protein kinase II (CaMKII). However, maintenance of tonic inhibition relied on protein kinase A activity. Depolarization of membrane potential (5 min of 0 mV holding) potentiated phasic inhibition via $Ca^{2+}$ and CaMKII but tonic inhibition was not affected. Thus, phasic and tonic inhibition seem to be independently maintained and regulated by different signaling cascades in the same cell. These results suggest that neuromodulatory signals might differentially regulate phasic and tonic inhibition in response to changes in brain states.

• BMB Reports
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• 제39권5호
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• pp.626-635
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• 2006

Lysophosphatidic acid acyltransferase (LPAAT) is an intrinsic membrane protein that catalyzes the synthesis of phosphatidic acid (PA) from lysophosphatidic acid (LPA). It is well known that LPAAT is involved in lipid biosynthesis, while its role in tumour progression has been of emerging interest in the last few years. To date, seven members of the LPAAT gene family have been found in human. Here we report a novel LPAAT member, designated as LPAAT-theta, which was 2728 base pairs in length and contained an open reading frame (ORF) encoding 434 amino acids. The LPAAT-theta gene consisted of 12 exons and 11 introns, and mapped to chromosome 4q21.23. LPAAT-theta was ubiquitously expressed in 18 human tissues by RT-PCR analysis. Subcellular localization of LPAAT-theta-EGFP fusion protein revealed that LPAAT-theta was distributed primarily in the endoplasmic reticulum (ER) of COS-7 cells. Furthermore, we found that the overexpression of LPAAT-theta can induce mTOR-dependent p70S6K phosphorylation on Thr389 and 4EBP1 phosphorylation on Ser65 in HEK293T cells.

• 대한바이러스학회지
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• 제28권1호
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• pp.53-62
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• 1998

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• 생명과학회지
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• 제26권3호
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• pp.282-288
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• 2016

단백질-단백질 결합은 수용체 단백질, 효소, 세포 골격 단백질의 세포내 위치 결정 및 기능 조절에 중요한 역할을 한다. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) 도메인을 가진 단백질들은 시냅스 가소성, 신경세포 성장과 분화뿐만 아니라 많은 질병의 병태생리에 중요하게 관여하는 scaffold 단백질로 작용한다. Multi-PDZ domain protein 1 (MUPP1)은 13개 PDZ 도메인을 가지는 단백질로서 세포막 수용체 군집화, 신호전달 복합체 구성, 세포 골격 조정에 대한 매개 역할을 하는 것으로 알려지고 있지만 MUPP1의 세포 내 기능은 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 아미노 말단 PDZ 도메인과 결합하는 새로운 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고 Wdpcp (전에 Fritz로 알려짐)이 MUPP1과 결합하는 것을 확인하였다. Wdpcp는 planar cell polarity (PCP) effector로서 세포 이동과 섬모형성에 관여하는 것으로 알려져 있다. Wdpcp는 MUPP1의 첫 번째 PDZ 도메인과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. 또한 MUPP1와 Wdpcp의 결합에서 Wdpcp의 C-말단부위가 결합에 필수적임을 효모 two-hybrid 방법으로 확인하였다. 이러한 단백질간 결합은 glutathione S-transferase (GST) pull-down assay, 공동면역침강, HEK-293T 세포에서의 발현위치를 통하여 추가적으로 확인하였다. 이러한 결과들은, MUPP1과 Wdpcp 결합은 세포내 액틴 다이내믹스(dynamics)와 세포이동 조절에 역할을 할 가능성을 시사한다.

• 생명과학회지
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• 제17권12호
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• pp.1627-1633
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• 2007

c-Jun N-terminal kinase (JNK)-interacting protein 1(JIP1)은 비계단백질(scaffold protein)로서 신경 세포와 ??장${\beta}$세포에서 많이 발현된다. 본 연구에서는 배양한 흰쥐 해마신경세포에서 JIP1, JIP-2 및 JIP-3을 모두 인식하는 항체를 이용하여 이들의 세포내 표현을 조사하였다. 전반적으로 JIP은 세포체와 가지돌기에 반점 모양으로 표현되었다. 이 JIP 반점들을 통계적으로 분석한 결과 흥분성 연접후표지인 PSD95 및 ${\alpha}CaMKII$ 반점 과 각각 $54.8{\pm}4.0%$ 및 $94.1{\pm}4.5%$가 겹쳐졌다. 반면에 억제성 연접후표지인 그리신수용체 및 gephyrin 반점과는 각각 단지 $8.6{\pm}0.5%$ 및 $7.3{\pm}0.5%$만 겹쳐졌다. 한편 lipid raft의 표지인 flotillin 반점의 상당부분$(29.3{\pm}1.0%)$이 JIP 반점과 겹쳐졌다. 또한, JIP은 일부 가지돌기의 끝부분에 매우 강하게 발편되었으며 축삭에는 표현이 미미하였다. 이 결과들은 JIP 단백질이 흥분성 연접후구역, 일부 lipid raft, 그리고 일부 가지돌기 끝부분에 주로 위치함을 의미한다.

• Parasites, Hosts and Diseases
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• 제54권6호
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• pp.725-732
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• 2016

Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, $PcyPHIST/CVC-81_{95}$, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about $PHIST/CVC-81_{95}$ protein in P. vivax. In this study, the recombinant $PvPHIST/CVC-81_{95}$ N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of $PvPHIST/CVC-81_{95}$ N and C termini in blood stage parasites was also determined. The antigenicity of recombinant $PvPHIST/CVC-81_{95}$ N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of $PvPHIST/CVC-81_{95}$ which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of $PvPHIST/CVC-81_{95}$. These results suggest that the $PvPHIST/CVC-81_{95}$ is localized on the CVCs and may be immunogenic in natural infection of P. vivax.

• Molecules and Cells
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• 제27권1호
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• pp.39-45
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• 2009

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain ($NOK{\Delta}ECD$) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be crosslinked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or $NOK{\Delta}ECD$ was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.

• Molecules and Cells
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• 제41권3호
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• pp.214-223
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• 2018

Oligoadenylate synthetase (OAS) protein family is the major interferon (IFN)-stimulated genes responsible for the activation of RNase L pathway upon viral infection. OAS-like (OASL) is also required for inhibition of viral growth in human cells, but the loss of one of its mouse homolog, OASL1, causes a severe defect in termination of type I interferon production. To further investigate the antiviral activity of OASL1, we examined its subcellular localization and regulatory roles in IFN production in the early and late stages of viral infection. We found OASL1, but not OASL2, formed stress granules trapping viral RNAs and promoted efficient RLR signaling in early stages of infection. Stress granule formation was dependent on RNA binding activity of OASL1. But in the late stages of infection, OASL1 interacted with IRF7 transcripts to inhibit translation resulting in down regulation of IFN production. These results implicate that OASL1 plays context dependent functions in the antiviral response for the clearance and resolution of viral infections.

• BMB Reports
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• 제37권4호
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• pp.402-407
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• 2004

A human gene T-complex protein 10 like (TCP10L) was cloned in our lab. A previous study showed that it expressed specifically in the liver and testis. A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity. In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen. This finding was confirmed by immunoprecipitation and subcellular localization experiments. As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells. Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.