NLRP3 Inflammasome Contributes to Lipopolysaccharide-induced Depressive-Like Behaviors via Indoleamine 2,3-dioxygenase Induction

Jeon, Seon-A,Lee, Eunju,Hwang, Inhwa,Han, Boyoung,Park, Sangjun,Son, Seunghwan,Yang, Jungmin,Hong, Sujeong,Kim, Chul Hoon,Son, Junghyun,Yu, Je-Wook Oxford University Press 2017 International Journal of Neuropsychopharmacology Vol.20 No.11

<P><B>Abstract</B></P><P><B>Background</B></P><P>Inflammation may play a significant role in the pathogenesis of depression, although the molecular target for the treatment of inflammation-mediated depressive symptoms remains to be elucidated. Recent studies have implicated the NLRP3 inflammasome in various psychiatric disorders, including depression. However, the underlying mechanism by which NLRP3 inflammasome activation mediates the progression of depressive-like behaviors remains poorly understood.</P><P><B>Methods</B></P><P>We examined whether NLRP3 deficiency influenced depressive-like behaviors and cerebral inflammation following systemic administration of lipopolysaccharide in mice. To further assess the contribution of the NLRP3 inflammasome to the progression of depression, we evaluated the effects of NLRP3 signaling on levels of indoleamine 2,3-dioxygenase.</P><P><B>Results</B></P><P><I>Nlrp3</I>-deficient mice exhibited significant attenuation of depressive-like behaviors and cerebral caspase-1 activation in a lipopolysaccharide-induced model of depression. Treatment with the antidepressant amitriptyline failed to block NLRP3-dependent activation of caspase-1, but inhibited lipopolysaccharide-promoted production of interleukin-1β mRNA via suppressing NF-κB signaling in mouse mixed glial cultures. Interestingly, lipopolysaccharide administration produced NLRP3-dependent increases in indoleamine 2,3-dioxygenase expression and activity of mouse brain. Furthermore, inflammasome-activating stimulations, but not treatment with the inflammasome product interleukin-1β, triggered <I>indoleamine 2,3-dioxygenase</I> mRNA induction in mixed glial cells.</P><P><B>Conclusions</B></P><P>Our data indicate that the NLRP3 inflammasome is significantly implicated in the progression of systemic inflammation-induced depression. NLRP3-dependent caspase-1 activation produced significant increases in indoleamine 2,3-dioxygenase levels, which may play a significant role in lipopolysaccharide-induced depression. Collectively, our findings suggest that indoleamine 2,3-dioxygenase is a potential downstream mediator of the NLRP3 inflammasome in inflammation-mediated depressive-like behaviors.</P>

pKT230 벡터를 이용한 Pseudomonas sp. P20 2,3-Dihydroxybiphenyl Dixygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

Biphenyl과 4-chlorobiphenyl을 분해하는 자연계 분리 균주인 Pseudomonas sp. P20의 chromosomal DNA로부터 pBluescript SK(+)를 이용하여 pcbABCD 유전자를 클로닝하여 재조합플라스미드 pCK1을 제조하였고, 또 pcbCD 유전자를 포함하여 pCK102을 제조하였다. 방향족 탄화수소 화합물의 생분해는 벤젠고리의 개환 과정이 중요하기 때문에, 2,3-dihydroxybiphenyl(2,3-DHBP)의 벤젠고리의 개환에 관여하는 2,3-DHBP dioxygenase(2,3-DHBD) 유전자를 pKT230 벡터를 이용하여 pCK102로부터 클로닝하였다. EcoRI으로 절단한 pCK102와 pKT230 벡터를 ligation시켜 13.8 kb의 hybrid plasmid pKK1을 제조하였다. 2,3-DHBD 유전자를 포함하는 pKK1을 E. coli XL1-Blue에 형질전환시켜 E. coli KK1 재조합 균주를 얻은 후, 2,3-DHBD의 활성을 측정하였다. E. coli KK1의 2,3-DHBD의 효소활성은 pBluescript SK(+)를 이용하여 제조한 재조합 균주인 E. coli CK102의 효소활성과 유사하였으나, Pseudomonas sp. DJ-12와 Pseudomonas sp. P20과 같은 자연계 분리균주보다 훨씬 높았다. Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2,3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2,3-DHBP dioxygenase activity. The specific 2,3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20

제조합균주 E. coli Ck1092가 생산하는 2,3-Dihydroxybiphenyl Dioxygenase의 정제 및 특성

박효남,김영수,김영창,김치경,임재윤 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3

4-CB 분해균주인 Pseudomonas sp. P20으로부터 pcbC 유전자를 클로닝하여 얻은 E. coli CK1092로부터 2,3-DHBP dioxygenase을 분리, 정제하여 효소적 특성을 조사하였다. 효소의 정제는 acetone 침전, DEAE-Se-phadex A-25 ion exchange chromatography, preparative electrophoresis 방법으로 정제하였다. 2,3-DHBP dioxygenase의 분자량은 약 270 kDa으로 추정되며, SDS-PAGE에 의한 분자량은 34 kDa이였다. 따라서, 동일한 subunit 8개 존재하는 octamer로 추정된다. 이 효소는 2,3-DHBP에 대해 높은 기질특이성을 보였으며, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol에 대해서는 활성을 보이지 않았다. 2,3-DHBP에 대한 Km 값은 18 μM이였으며 30μM 이상의 기질농도에서 활성이 감소하였다. 효소활성의 최적 pH는 8.0이였으며, pH 7.0~10.0 범위에서는 안정하였고, 최적 활성 온도는 40~60℃이며, 60℃까지는 비교적 안정하였다. 또한, 이 효소는 Cu^2+, Fe^2+, Fe^3+ 이온들에 의하여 효소활성이 저해되었고, H_2O_2와 EDTA에 의해서도 활성이 저해되었으며, 10%의 유기용매에 의해서 안정화되지 않았다. 효소활성부위를 알아보기 위해 화학변형제를 처리해 본 결과 tryosine, tryptophan과 histidine이 효소활성에 관여할 것으로 추정된다. 2,3-DHBP dioxygenase was purified from E. coli CK1092 carrying the pcbC gene, which was cloned from 4-chlorobiphenyl-degrading Pseudomonas sp. P20. Purification of this enzyme was done by acetone precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and preparative gel electrophoresis. The molecular weight of subunit was 34 kDa determined by SDS-PAGE, and that of native enzyme was about 270 kDa. It suggests that this enzyme consist of eight identical subunits. This enzyme was specifically active against only 2,3-DHBP as a substrate with 18 μM of Km value, but not catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol. The optimal pH and temperature of 2,3-DHBP dioxygenase were pH 8.0 and 40~60℃. The enzyme was inhibited by Cu^2+, Fe^2+ and Fe^3+ ions, and was inactivated by H_2O_2 and EDTA. The lower concentrations of some organic solvents such as acetone and ethanol don't stabilize the activity of 2,3-DHBP dioxygenase. The enzyme was completely inactivated by adding the reagents such as N-bromosuccinimide, iodine and p-diazobenzene sulfonic acid.

Pseudomonas sp. DJ-12에서 분리한 2,3-Dihydroxybiphenyl Dioxygenase의 효소학적 특성

성태경,남정현,김치경 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.2

Pseudomonas sp. DJ-12로부터 2,3-DHBP dioxygease를 아세톤 침전, DEAE- Sephadex A-50 ion exchange chromatograpy, Sephadex G-150 gel filtration chromatography 방법으로 분리정제하였다. 이 효소의 분자량은 약 260 kD였으며, SDS-PAGE를 수행한 결과 33 kD의 subunit 8개로 이루어진 octamer임을 알 수 있었다. 2,3-DHBP에 대한 이 효소의 K_m값은 약 61 μM이었다. 이 효소의 최적 활성온도는 20∼70℃ 였으며, 10∼50℃의 온도에서 안정하였다. 또한 이 효소의 최적 pH는 8.0이었으며 pH 6.0∼8.0에서 안정성을 나타내었다. Catechol에 대한 2,3-DHBP dioxygenase의 활성도는 2,3-DHBP에 비하여 약 45%였으나, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol의 기질에 대해서는 특이성이 거의 없었다. The 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase, the product of pcbC gene, was purified from the biphenyl and 4-chlorobiphenyl degrading Pseudomonas sp. DJ-12 by the methods of acetone precipitation, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-150 gel filtration chromatography. The enzyme was estimated to be about 260 kilodaltons in molecular weight and to be consisted of eight subunits. The K_m value of thhe enzyme was 61 μM to 2,3-DHBP and the highest activity of the enzyme was observed at pH 8 and 30℃. The 2,3-DHBP dioxygenase showed enzymatic activity to the substrate of catechol, but not to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol.

Aniline 분해세균 Delftia sp. JK-2에서 분리된 catechol 2,3-dioxygenase의 특성 및 N-말단 아미노산 서열분석

황선영,송승열,오계헌 한국미생물학회 2003 미생물학회지 Vol.39 No.1

The aim of this work was to investigate the characterization and sequence of catechol 2,3-dioxygenase isolated from Delfia sp. JK-2, which could utilize aniline as sole carbon, nitrogen and energy source. In initial experiments, several characteristics of C2,3O separated with ammonium sulfate precipitation, DEAE-sepharose were investigated. Specific activity of C2,3O was approximately 4.72 unit/mg. C2,3O demonstrated its enzyme activity to other substrates, catechol and 4-methylcatechol. The optimum temperature of C2,3O was $$Cu^{2+}$^{\circ}C$, and the optimal pH was approximately 8. Metal ions such as $Ag^{+}$, $Hg^{+}$, and $Cu^{2+}$ showed inhibitory effect on the activity of C2,3O. Molecular weight of the enzyme was determined to approximately 35 kDa by SDS-PAGE. N-terminal amino acid sequence of C2,3O was analyzed as $^{1}MGVMRIG-HASLKVMDMDA- AVRHYENV^{26}$, and exhibited high sequence homology with that of C2,30 from Pseudomonas sp. AW-2, Comamonas sp. JS765, Comamonas testosteroni and Burkholderia sp. RPO07. PCR product was amplified with the primers derived from N-terminal amino acid sequence. In this work, we found that the amino acid sequence of Delftia sp. JK-2 showed high sequence homology of C2,3O from Pseudomonas sp. AW-2 (100%) and Comamonas sp. JS765 (97%).

Purification and Characterization of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707

심현우,조용권,정미자 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.4

Protocatechuate 3,4-dioxygenase was isolated and characterized from Pseudomonas pseudoalcaligenes KF707 for the purpose of developing a new anti-browning agent. The protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707 was purified 296.8-fold, and showed specific activity of 121.7 U/mg. Based on the SDS-polyacrylamide and gel permeation chromatography, the molecular weight of protocatechuate 3,4-dioxygenase was 189.9 kDa and was composed of 3 αβ protomers,with molecular weights of 29.0 kDa of α subunit and 34.3 kDa of â subunit. The optimal pH and temperature were 7.5 and 38oC,respectively. Km values of catechol, protocatechuate, gallate, pcresol,caffeic acid, catechin, L-DOPA, 4-methylcatechol and pyrogallol were 14, 17, 2, 10, 12, 20, 30, 21 and 3 μM, and the Vmax/Km (mim−1) values were 0.052, 3.06, 0.35, 0.01, 0.03, 0.02,0.006, 0.008 and 0.11, respectively. This indicates that the enzyme is active on a wide range of phenyl compounds, in contrast to the high specificity of similar enzymes from other sources. Our data also show that the turnover number of protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707 is 68s−1, which is much higher than the known values from other sources.

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression

Noh, Kyung Tae,Son, Kwang Hee,Jung, In Duk,Kang, Tae Heung,Choi, Chang Hun,Park, Yeong-Min American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.19

<P>Background: IDO functions as a crucial mediator of tumor-mediated immune tolerance. Results: Here, we found that GSK-3-dependent IDO expression in the dendritic cell plays a role in anti-tumor activity via the regulation of CD8(+) T-cell proliferation and CTL activity. Conclusion: GSK-3 activity functions in the immune enhancement-mediated anti-tumor response via IDO regulation. Significance: We proved the effectiveness of the GSK-3-dependent IDO regulatory mechanism in DC-based cancer vaccination. Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3 (GSK-3) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase C (PKC) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3 inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)--induced IDO expression via GSK-3 activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN--induced activation of GSK-3. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN--induced activation of GSK-3 in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon--induced IDO expression via GSK-3 activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.</P>

트립토판 대사체 3-hydroxyanthranilic acid 의 TRAIL-유도 활성 T 세포 사멸 효과

Su-Kil Seo(서수길) 한국생명과학회 2011 생명과학회지 Vol.21 No.2

Indoleamine 2,3-dioxygenase (IDO)에 의한 트립토판 대사체의 생성은 T 세포에 강력한 억제효과를 미치지만 여전히 그 작용기전에 대한 연구보고는 미비한 실정이다. 본 연구자는 트립토판 대사체 3-HAA가 선택적으로 활성 T 세포의 사멸을 촉진시키는 효과가 있음을 확인하였고, 이는 세포주기 억제와는 관련이 없었다. 3-HAA처리 시 활성 T 세포에서 TRAIL과 그의 수용체의 발현이 현저히 증가하고, 이들 상호작용을 차단하였을 때 3-HAA-매개 활성 T 세포사멸 효과가 유의하게 낮아졌다. 본 연구를 통해 트립토판 대사체 3-HAA의 선택적 T세포 억제 효과가 TRAIL-유도 세포사멸과 관련됨을 알 수 있다. Generation of tryptophan-derived metabolites by indoleamine 2,3-dioxygenase (IDO) is a potent immunoregulatory mechanism in T cell responses. However, the mechanism remains unclear. We showed that 3-hydroxyanthranilic acid (3-HAA), the most potent metabolite, selectively induced apoptosis in activated T cells, but not in resting T cells. This was not associated with cell cycle arrest. We found that TRAIL expression was selectively induced in activated T cells by treatment of 3-HAA. Blockade of the TRAIL: DR4/DR5 pathway significantly inhibited 3-HAA-mediated T cell death. Our data suggest that TRAIL-induced apoptosis is involved in the mechanism of 3-HAA-mediated T cell death.

Characterization of an Catechol 2,3-dioxygenase from Pseudomonas putida SU10

Ha, You Mee,Min, Kyung Hee,Jung, Young Hee 숙명여자대학교 자연과학연구소 1998 자연과학논문집 Vol.- No.9

Pseudomonas putida SUl0의 4-methylcatechol 2,3-dioxygenase의 정제를 ammonium sulfate 침전, DEAE 5PW, superdex S-200, Resource-Q의 chromatog chromatography를 통하여 실시하였다. Gel filtration 에 의한 분자량을 약 130 kDa이며 SDS/polyacrylamide gel electrophoresis에 의한 subunit의 분자량은 34 kDa이므로 이 효소는 4개의 동일한 subunit로 되어 있음을 확이하였다. 이 효소의 N-terminal amino acid 서열을 결정한 결과 다음 extradiol dioxygenases와 높은 동질성을 보여주었다. Catechol과 methyl로 치환된 catechol에 대한 활성을 조사한 결과 catechol이나 3-methylcatechol보다 4-methylcatechol에 대하여 높게 나타내었다. 이들 기질에 대한 Km 값은 3.5-5.07μM 이다. A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU1O by a purification procedure consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicates a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit 34 kDa by SDS/ polyacrylamide gel electrophoresis. These results suggest that the native enzyme was composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identify with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. Km values for these substrates with this enzyme are around 3.5-5.7μM.

Inhibition and Chemical Mechanism of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707

Taekyeong Kang(강태경),Sang Ho Kim(김상호),Mi Ja Jung(정미자),Yong Kweon Cho(조용권) 한국생명과학회 2015 생명과학회지 Vol.25 No.5

Pseudomonas pseudoalcaligenes KF707에서 정제한 protocatechuate 3,4-dioxygenase의 특징을 조사하기 위하여 pH안정성, 화학적 저해, 화학적 수식과 pH의존성 반응 상수에 대한 실험을 수행하였다. 이 효소는 pH 4.5~10.7에서 안정하였다. L-ascorbate와 glutathione은 Kis가 각각 0.17 mM과 0.86 mM인 경쟁적 저해제였으며, DL-dithiothreitol은 Kis 1.57 mM 및 Kii 8.08 mM의 비경쟁적 저해패턴을 나타내었다. Potassium cyanide, p-hydroxybenzoate 및 sodium azide는 Kis가 각각 55.7 mM, 0.22 mM 및15.64 mM이었으며, Kii는 각각94.1 mM, 8.08 mM, 및 662.64 mM인 비경쟁적 저해패턴을 나타내었다. FeCl2는 Kis가 29 μM로 가장 우수한 경쟁적 저해제였으며, FeCl3, MnCl2, CoCl2, HgCl2, AlCl3도 각각 Kis가 1.21 mM, 0.85 mM, 3.98 mM, 0.17 mM 및 0.21 mM인 경쟁적 저해패턴을 보였다. 한편, 다른 금속이온들은 비경쟁적 저해패턴을 나타내었다. pH의존성 반응상수의 실험결과로부터 pK 6.2와 9.4의 촉매부위와 pK 5.5와 9.0의 결합부위가 존재함을 알 수 있었다. Lysine, cysteine, tyrosine, carboxyl과 histidine은 각각의 고유한 화학적 수식제에 의해 수식되었는데, 이는 이들 잔기들이 결합과 촉매에 관여한다는 것을 나타낸다. 위 결과를 토대로 화학적 메커니즘을 제시한다. We carried out pH stability, chemical inhibition, chemical modification, and pH-dependent kinetic parameter assessments to further characterize protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. Protocatechuate 3,4-dioxygenase was stable in the pH range of 4.5~10.5. L-ascorbate and glutathione were competitive inhibitors with Kis values of 0.17 mM and 0.86 mM, respectively. DL-dithiothreitol was a noncompetitive inhibitor with a Kis value of 1.57 mM and a Kii value of 8.08 mM. Potassium cyanide, p-hydroxybenzoate, and sodium azide showed a noncompetitive inhibition pattern with Kis values of 55.7 mM, 0.22 mM, and 15.64 mM, and Kii values of 94.1 mM, 8.08 mM, and 662.64 mM, respectively. FeCl2 was the best competitive inhibitor with a Kis value of 29 μM. FeCl3, MnCl2, CoCl2, and AlCl3 were also competitive inhibitors with Kis values of 1.21 mM, 0.85 mM, 3.98 mM, and 0.21 mM, respectively. Other metal ions showed noncompetitive inhibition patterns. The pH-dependent kinetic parameter data showed that there may be at least two catalytic groups with pK values of 6.2 and 9.4 and two binding groups with pK values of 5.5 and 9.0. Lysine, cysteine, tyrosine, carboxyl, and histidine were modified by their own specific chemical modifiers, indicating that they are involved in substrate binding and catalysis.