Re: 산화질소 : 미토콘드리아 신생합성, 소포체 스트레스 제거 .. nature review

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• Mini Review
• Published: 01 January 2005

Minireview

Nitric oxide:

orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response

• Weiming XU, 
• Ian G CHARLES & 
• Salvador MONCADA 

Cell Research volume 15, pages63–65 (2005)Cite this article

•  
•  

ABSTRACT

Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations, the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of “metabolic hypoxia” – a situation in which, although oxygen is available, the cell is unable to utilise it.

미토콘드리아는 

산화적 인산화 과정을 통해 

ATP 분자 형태로 에너지를 생성하는 

살아있는 세포의 발전소로 오랫동안 여겨져 왔습니다. 

지난 20년 동안 

미토콘드리아가 세포 사멸, 

즉 프로그램된 세포 사멸을 실행하는 데 중요한 역할을 한다는 사실이 밝혀졌습니다. 

최근에는 

미토콘드리아가 

세포 방어 반응  cellular defence responses의 조율에도 참여한다는 사실이 분명해졌습니다. 

생리학적 농도에서 

기체 분자 산화질소(NO)는 

산소와 경쟁하여 

미토콘드리아 효소인 시토크롬 C 산화효소(복합체 IV)를 

억제합니다. 

이러한 

상호작용은 

미토콘드리아 신호 전달을 통한 

세포 호흡의 생리적 조절부터 

산소를 사용할 수 있지만 

세포가 이를 활용하지 못하는 상황인 '대사성 저산소증'의 발생에 이르기까지 

NO의 미토콘드리아 작용의 근간을 이룹니다.

https://www.ahajournals.org/doi/10.1161/ATVBAHA.108.181628?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

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NO AND MITOCHONDRIAL RESPIRATION

Nitric oxide (NO) plays an important role as an inter- and intra-cellular messenger, acting in the cardiovascular system, in the nervous system and as a component of the immune system 1. NO is a diatomic free radical which is a gas at room temperature, making it highly diffusible within the vasculature. One of its major targets is the soluble guanylyl cyclase (sGC). The binding of NO to Fe2+ in the haem group of sGC activates the enzyme and increases the concentration of cGMP (guanosine 3', 5'-cyclic monophosphate). The NO/cGMP signalling pathway has been demonstrated to be important in many biological systems including blood pressure regulation, platelet aggregability, smooth muscle relaxation and peripheral and central neurotransmission.

Recent work has shown that NO, in the nanomolar concentration range that activates sGC, can also bind to cytochrome c oxidase (complex IV) and inhibit it in a manner that is reversible and in competition with oxygen 2, 3. Cytochrome c oxidase is the terminal enzyme of the mitochondrial respiratory chain and is responsible for 90% of cellular oxygen consumption in mammals. The enzyme catalyses the oxidation of cytochrome c2+ to cytochrome c3+ and the reduction of oxygen to water, which is coupled to the pumping of protons across the inner mitochondrial membrane. Because NO competes with oxygen at the cytochrome c oxidase, increases in NO concentration can prevent the enzyme from using any available oxygen, thus causing a kind of “metabolic hypoxia” 4.

Such “metabolic hypoxia” has profound biological consequences, particularly with regard to the regulation of the hypoxia-inducible factor (HIF)-1. HIF-1 is a transcription factor that plays a major role in the response of tissues to low partial pressures of oxygen, by upregulating genes such as those involved in glycolysis and angiogenesis 5. At normal oxygen concentrations HIF-1 is regulated in an oxygen-dependent manner by a family of prolyl hydroxylases that prevent stabilisation of HIF-1α its subsequent dimerisation with HIF-1β and the consequent activation of target genes. At low oxygen concentrations (1-3%) prolyl hydroxylase activity is inhibited, and HIF-1α accumulates, resulting in the activation of target gene expression‎. However, if cells maintained at 1-3% oxygen are exposed to low concentrations of NO, stabilisation of HIF-1α is prevented 6. We have shown that, in hypoxic conditions, NO and other inhibitors of mitochondrial respiration can cause redistribution of oxygen away from the electron transport chain towards non-respiratory oxygen-dependent targets such as prolyl hydroxylases 7. Since more than 70 genes (including those involved in erythropoiesis, angiogenesis and glycolysis) are known to be regulated at the transcriptional level by HIF-1 5, NO-mediated inhibition of respiration may profoundly modify the signalling consequences of hypoxia.

산화질소(NO)는 

심혈관계, 신경계 및 면역 체계의 구성 요소로 작용하는 

세포 간 및 세포 내 메신저로서 중요한 역할을 합니다 1. 

NO는 

상온에서 기체인 이원자 자유 라디칼 diatomic free radical 로 

혈관계 내에서 확산성이 매우 높습니다. 

주요 표적 중 하나는 

수용성 구아닐 시클라제(sGC)입니다.  

soluble guanylyl cyclase (sGC)

sGC의 헤모 그룹에서 

NO가 Fe2+에 결합하면 

효소가 활성화되고 

cGMP(구아노신 3', 5'-사이클릭 모노포스페이트)의 농도가 증가합니다. 

참고) 

cGMP is a common regulator of ion channel conductance, glycogenolysis, and cellular apoptosis. It also relaxes smooth muscle tissues. In blood vessels, relaxation of vascular smooth muscles leads to vasodilation and increased blood flow. At presynaptic terminals in the striatum, cGMP controls the efficacy of neurotransmitter release.[5]

cGMP is a secondary messenger in phototransduction in the eye. In the photoreceptors of the mammalian eye, the presence of light activates phosphodiesterase, which degrades cGMP. The sodium ion channels in photoreceptors are cGMP-gated, so degradation of cGMP causes sodium channels to close, which leads to the hyperpolarization of the photoreceptor's plasma membrane and ultimately to visual information being sent to the brain.[6]

cGMP is also seen to mediate the switching on of the attraction of apical dendrites of pyramidal cells in cortical layer V towards semaphorin-3A (Sema3a).[7] Whereas the axons of pyramidal cells are repelled by Sema3a, the apical dendrites are attracted to it. The attraction is mediated by the increased levels of soluble guanylate cyclase (SGC) that are present in the apical dendrites. SGC generates cGMP, leading to a sequence of chemical activations that result in the attraction towards Sema3a. The absence of SGC in the axon causes the repulsion from Sema3a. This strategy ensures the structural polarization of pyramidal neurons and takes place in embryonic development.

cGMP, like cAMP, gets synthesized when olfactory receptors receive odorous input. cGMP is produced slowly and has a more sustained life than cAMP, which has implicated it in long-term cellular responses to odor stimulation, such as long-term potentiation. cGMP in the olfactory is synthesized by both membrane guanylyl cyclase (mGC) as well as soluble guanylyl cyclase (sGC). Studies have found that cGMP synthesis in the olfactory is due to sGC activation by nitric oxide, a neurotransmitter. cGMP also requires increased intracellular levels of cAMP and the link between the two second messengers appears to be due to rising intracellular calcium levels.[8]

cGMP는 

이온 채널 전도도, 

글리코겐 분해 및 

세포 세포 사멸을 조절하는 일반적인 조절제입니다. 

또한 평활근 조직을 이완시킵니다. 

혈관에서 

혈관 평활근의 이완은 

혈관 확장 및 혈류 증가로 이어집니다. 

선조체의 시냅스 전 단자에서 c

GMP는 신경전달물질 방출의 효능을 조절합니다[5].

cGMP는 

눈의 광전달에서 

보조 메신저 역할을 합니다. 

포유류 눈의 광수용체에서 빛의 존재는 포스포디에스테라아제를 활성화하여 cGMP를 분해합니다. 광수용체의 나트륨 이온 채널은 cGMP-게이팅되어 있으므로 cGMP가 분해되면 나트륨 채널이 닫히고, 이는 광수용체 원형막의 과분극으로 이어져 궁극적으로 시각 정보가 뇌로 전송됩니다[6].

cGMP는 또한 

피질 V 층에 있는 피라미드 세포의 정단 수상 돌기가 

세마포린-3A(Sema3a)로 끌어당기는 것을 매개하는 것으로 보입니다.[7] 피라미드 세포의 축삭은 세마3a에 의해 밀려나는 반면, 정단 수상 돌기는 끌어당겨집니다. 이러한 인력은 정단 수상 돌기에 존재하는 수용성 구아닐레이트 시클라제(SGC)의 증가된 수준에 의해 매개됩니다. SGC는 cGMP를 생성하여 일련의 화학적 활성화로 이어져 Sema3a로 끌어당기는 결과를 낳습니다. 축삭에 SGC가 없으면 Sema3a로부터 반발이 일어납니다. 이 전략은 피라미드 뉴런의 구조적 분극을 보장하고 배아 발달에서 일어납니다.

후각 수용체가 냄새 입력을 받으면 cAMP와 마찬가지로 cGMP가 합성됩니다. cGMP는 천천히 생성되며, 장기 강화와 같은 냄새 자극에 대한 장기적인 세포 반응에 관여하는 cAMP보다 더 오래 지속됩니다. 후각의 cGMP는 막 구닐 시클라제(mGC)와 용해성 구닐 시클라제(sGC) 모두에 의해 합성됩니다. 연구에 따르면 후각에서 cGMP 합성은 신경전달물질인 산화질소에 의한 sGC 활성화로 인한 것으로 나타났습니다. 또한 cGMP는 세포 내 cAMP 수치가 증가해야 하며, 두 번째 메신저 사이의 연결은 세포 내 칼슘 수치 상승으로 인한 것으로 보입니다[8].

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9827765/

NO/cGMP 신호 경로는 

혈압 조절, 

혈소판 응집성, 

평활근 이완, 

말초 및 중추 신경 전달 등 

많은 생물학적 시스템에서 중요한 역할을 하는 것으로 입증되었습니다.

최근 연구에 따르면 

sGC를 활성화하는 나노몰 농도 범위에서 

NO는 

시토크롬 c 산화효소(복합체 IV)에 결합하여 

가역적이고 산소 2, 3과 경쟁하는 방식으로 

이를 억제할 수 있는 것으로 나타났습니다. 

시토크롬 c 산화효소는 

미토콘드리아 호흡 사슬의 말단 효소이며 

포유류에서 세포 산소 소비의 90%를 담당합니다. 

시토크롬 c 산화효소

시토크롬 c2+가 시토크롬 c3+로 산화되고 

산소가 물로 환원되는 과정을 촉매하며, 

이는 미토콘드리아 내부 막에서 양성자가 펌핑되는 것과 결합되어 있습니다. 

NO는 

시토크롬 c 산화 효소에서 

산소와 경쟁하기 때문에 

NO 농도가 증가하면 효소가 사용 가능한 산소를 사용하지 못하게 되어 

일종의 "대사성 저산소증"4을 유발할 수 있습니다.

이러한 "대사성 저산소증"은 

특히 저산소증 유도 인자(HIF)-1의 조절과 관련하여 

생물학적으로 중대한 결과를 초래합니다. 

HIF-1은 

당분해 및 

혈관 신생에 관여하는 유전자 등의 유전자를 상향 조절하여 

낮은 산소 분압에 대한 조직의 반응에 중요한 역할을 하는 

전사인자입니다. 

정상적인 산소 농도에서 

HIF-1은 

산소 의존적인 방식으로 조절되며, 

이는 HIF-1α의 안정화를 방지하는 프롤릴 하이드 록실라 제 계열에 의해 

HIF-1β와의 이합체화 및 

그에 따른 목표 유전자의 활성화를 방지합니다. 

낮은 산소 농도(1~3%)에서는 

프롤릴 하이드 록실 라제 활성이 억제되고 

HIF-1α가 축적되어 

표적 유전자 발현이 활성화됩니다. 

그러나 

1~3%의 산소 농도를 유지하는 세포가 

저농도의 NO에 노출되면 

HIF-1α의 안정화가 방지됩니다. 

우리는 

저산소 조건에서 

NO 및 

기타 미토콘드리아 호흡 억제제가 

전자 수송 사슬에서 프롤릴 하이드 록실 라제와 같은 

비호흡 산소 의존 표적 7로 산소를 재분배할 수 있음을 보여주었습니다. 

70개 이상의 유전자(적혈구 생성, 혈관 신생 및 해당 작용에 관여하는 유전자 포함)가 

HIF-1에 의해 전사 수준에서 조절되는 것으로 알려져 있으므로 5, 

NO 매개 호흡 억제는 

저산소증의 신호 결과를 크게 수정할 수 있습니다.

NO AND THE ENDOPLASMIC RETICULUM STRESS RESPONSE

Until recently, the main function of mitochondria was considered to be the production of energy; however mitochondria can also play an important role in intracellular signalling, for example by coupling calcium signalling with the endoplasmic reticulum (ER) 8, 9, 10. Studies using genetically targeted Ca2+-selective probes (such as the bioluminescent protein aequorin) or fluorescent dyes (such as rhod-2) have provided a direct and unambiguous readout of mitochondrial Ca2+ changes in living cells following receptor stimulation. Recently we found that in NO-generating cells there is a disruption in the respiratory chain and a decreased respiration rate that is accompanied by a mitochondrial Ca2+ flux 11. These findings are in agreement with previous reports showing that exogenous NO 12 and peroxynitrite 13, 14 can result in the release of Ca2+ from mitochondria. Interestingly, Nowicky and Duchen reported changes in [Ca2+]i and membrane currents when mitochondrial respiration in dissociated rat hippocampal neurons was impaired by inhibition of cytochrome c oxidase using cyanide, or by hypoxia 15. It is possible that disrupting electron transfer at cytochrome c oxidase may result in ionic readjustment and modulation of the Ca2+ flux between the mitochondria and the ER. We found that this NO-mediated change in Ca2+ flux is sufficient to activate the Ca2+-dependent protease (site-1 protease, S1P) involved in a regulated intermembrane proteolysis (RIP) pathway. S1P, in association with the site-2 protease located in the Golgi apparatus, cleaves the ER stress-regulated transmembrane transcription factor p90 ATF6. The resulting soluble transcription factor p50 ATF6 is then free to translocate to the nucleus where it subsequently activates ER stress-responsive genes, such as glucose regulated protein 78 (Grp78) 11.

Grp78 is a Ca2+-binding chaperone protein with cytoprotective properties. It can be up-regulated by hypoxic conditions and by glucose starvation. Grp78 associates transiently with nascent proteins, facilitating their translocation into the ER and aiding in their folding and transport through the ER. Grp78 appears to be a master switch of the unfolded protein response (UPR) and ER stress. We found that the NO-mediated up-regulation of Grp78 provides significant cytoprotection against toxic agents including thapsigargin (a selective ER Ca2+-ATPase inhibitor). This cytoprotection is abolished following treatment with cyclosporin A (CsA), which prevents mitochondrial Ca2+ efflux, and BAPTA-AM, an intracellular chelator. The NO-mediated ER stress response is diminished in rho0 cells that are devoid of mitochondrial DNA. Therefore, our work has shown that NO signals, via mitochondrial respiration, to the ER stress response with consequences for cell survival or death 11.

Apart from Grp78, a number of other genes have been shown to be regulated by the Ca2+-dependent serine protease S1P. These include genes for growth factors, transcription factors and viral glycoproteins that are involved in neurological, cardiovascular and immunological systems. Our findings suggest that the NO-mediated Ca2+ signalling pathway may well be important in their regulation.

최근까지 미토콘드리아의 주요 기능은 

에너지 생산으로 간주되었지만, 

미토콘드리아는 

칼슘 신호 전달을 소포체(ER) 8, 9, 10와 결합하여 

세포 내 신호 전달에도 중요한 역할을 할 수 있습니다. 

유전자 표적 Ca2+ 선택적 프로브(예: 생체발광 단백질 아에코린) 또는 

형광 염료(예: rhod-2)를 사용한 연구는 

수용체 자극 후 살아있는 세포의 미토콘드리아 Ca2+ 변화를 

직접적이고 명확하게 판독할 수 있는 방법을 제공했습니다. 

최근에 우리는

 NO 생성 세포에서 

호흡 사슬이 중단되고 

미토콘드리아 Ca2+ 플럭스 11가 동반되는 

호흡 속도가 감소한다는 사실을 발견했습니다. 

이러한 결과는 

외인성 NO 12 및 

퍼옥시니트라이트 13, 14가 

미토콘드리아에서 Ca2+를 방출할 수 있다는 이전 보고와 일치합니다. 

흥미롭게도 이제키와 듀첸은 

해리된 쥐 해마 뉴런의 미토콘드리아 호흡이 

시안화물을 사용한 사이토크롬 c 산화효소의 억제 또는 

저산소증에 의해 손상되었을 때 

[Ca2+]i 및 막 전류의 변화를 보고했습니다15. 

사이토크롬 c 산화효소에서 

전자 전달을 방해하면 

미토콘드리아와 ER 사이의 이온 재조정 및 

Ca2+ 플럭스의 조절이 발생할 수 있다는 가능성이 있을 수 있습니다. 

우리는 이러한 NO 매개 Ca2+ 플럭스의 변화가 

조절된 막간 단백질 분해(RIP) 경로에 관여하는 

Ca2+ 의존성 프로테아제(사이트-1 프로테아제, S1P)를 활성화하기에 

충분하다는 것을 발견했습니다. 

S1P는 

골지체에 위치한 사이트-2 프로테아제와 연계하여 

ER 스트레스 조절 막간 전사인자 p90 ATF6을 절단합니다. 

그 결과 

생성된 수용성 전사 인자 p50 ATF6는 

핵으로 자유롭게 이동하여 

포도당 조절 단백질 78(Grp78) 11과 같은 

ER 스트레스 반응 유전자를 활성화합니다.

https://www.nature.com/articles/cdd2010104

스핑고지질 대사산물인 세라마이드와 스핑고신-1-인산염(S1P)은 

최근 오토파지에 관여하는 것으로 밝혀졌습니다. 

이 연구에서는 

S1P를 특이적으로 탈인산화하는 

소포체(ER)에 존재하는 효소인 스핑고신-1-포스페이트 포스포하이드롤라제-1(SPP1)의 결핍이 

자가포식을 유도한다는 사실을 보고했습니다. 

라파마이신의 포유류 표적과 클

래스 III 포스포이노시타이드 3-키나아제/베클린-1 경로는 관여하지 않았고 

이 자가포식은 p53과 독립적이었지만, 

SPP1 결핍 후 C/EBP 동족 단백질, BiP, 인산화 유핵 번역 개시 인자-2α, 

ER 스트레스 하류 표적인 프로카스파제 2와 4의 절단이 증가했습니다. 

자가포식은 

RNA 유사 ER 키나아제(PERK), 

이노시톨 요구형 트랜스막 키나아제/엔도뉴클레아제-1α에 의해 조절되는 단백질 키나아제의 고갈 또는 

ER 스트레스에 대한 전개 단백질 반응(UPR)의 

3가지 센서인 전사인자 6의 활성화에 의해 억제되었습니다. 

SPP1의 하향 조절에 의해 촉발된 자가포식은 

세포 사멸로 이어지지 않고 

오히려 PERK에 의존적인 방식으로 생존 신호인 Akt를 자극하여 

이를 억제하면 세포가 세포 사멸에 민감하게 반응합니다. 

SPP1의 결핍은 

S1P의 세포 내 수준과 분비를 증가시켰지만 

세포 표면 S1P 수용체의 활성화는 오토파지를 유도하지 않았습니다. 

그럼에도 불구하고, 

S1P의 세포 내 풀이 증가했지만 

디하이드로-S1P는 증가하지 않았으며 

자가포식 및 ER 스트레스를 유도했습니다. 

따라서 

SPP1은 

세포 내 S1P 항상성을 조절함으로써 

UPR 및 ER 스트레스에 의한 자가포식을 조절할 수 있습니다.

Grp78은 

세포 보호 특성을 가진 

Ca2+ 결합 샤프론 단백질입니다. 

저산소 상태와 

포도당 결핍에 의해 상향 조절될 수 있습니다. 

Grp78은 

초기 단백질과 일시적으로 결합하여 

ER로의 전위를 촉진하고 

ER을 통한 단백질의 접힘과 수송을 돕습니다. 

Grp78은 

펼쳐진 단백질 반응(UPR)과 

ER 스트레스의 마스터 스위치 역할을 하는 것으로 보입니다. 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094232/

우리는 

Grp78의 NO 매개 상향 조절이 

탭시가르긴(선택적 ER Ca2+-ATPase 억제제)을 포함한 독성 물질에 대해 

상당한 세포 보호 기능을 제공한다는 사실을 발견했습니다. 

이러한 

세포 보호 효과는 

미토콘드리아 Ca2+ 유출을 방지하는 

사이클로스포린 A(CsA)와 

세포 내 킬레이트제인 BAPTA-AM으로 치료하면 사라집니다. 

NO 매개 ER 스트레스 반응은 

미토콘드리아 DNA가 없는 rho0 세포에서 감소합니다. 

따라서 

우리의 연구는 

NO가 

미토콘드리아 호흡을 통해 

ER 스트레스 반응에 신호를 보내 

세포 생존 또는 사멸에 영향을 미친다는 것을 보여주었습니다 11.

Grp78 외에도 

많은 다른 유전자들이 

Ca2+ 의존성 세린 프로테아제 S1P에 의해 조절되는 것으로 나타났습니다. 

여기에는 

신경계, 

심혈관계 및 면역계에 관여하는 성장 인자,

 전사인자 및 바이러스 당단백질에 대한 유전자가 포함됩니다. 

우리의 연구 결과는 

NO 매개 Ca2+ 신호 전달 경로가 

이들의 조절에 중요할 수 있음을 시사합니다.

CONCLUSION

The coupling of mitochondrial signalling and the ER is an emerging area of interest, and a common apoptosis pathway has recently been described in BAX and BAK regulation in which the ER and mitochondria are coupled via Ca2+ 16. It will be important to explore further how the NO-mediated ER signalling we have described interacts with BAX and BAK pathways to regulate mitochondria-mediated apoptosis.

미토콘드리아 신호와

 ER의 결합은 새로운 관심 분야이며, 

최근 ER과 미토콘드리아가 

Ca2+ 16을 통해 결합하는 BAX 및 BAK 조절에서 

일반적인 세포 사멸 경로가 설명되었습니다. 

우리가 설명한 

NO 매개 ER 신호가 

미토콘드리아 매개 세포 사멸을 조절하기 위해 

BAX 및 BAK 경로와 어떻게 상호 작용하는지 

더 탐구하는 것이 중요할 것입니다.

References

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