CD38作为细胞 NAD 调节因子的研究进展

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CD38 as a Regulator of Cellular NAD: A Novel Potential Pharmacological Target for Metabolic Conditions

Abstract

摘要

CD38 is a multifunctional enzyme that uses nicotinamide adenine dinucleotide (NAD) as a substrate to generate second messengers. Recently, CD38 was also identified as one of the main cellular NADases in mammalian tissues and appears to regulate cellular levels of NAD in multiple tissues and cells. Due to the emerging role of NAD as a key molecule in multiple signaling pathways, and metabolic conditions it is imperative to determine the cellular mechanisms that regulate the synthesis and degradation of this nucleotide. In fact, recently it has been shown that NAD participates in multiple physiological processes such as insulin secretion, control of energy metabolism, neuronal and cardiac cell survival, airway constriction, asthma, aging and longevity. The discovery of CD38 as the main cellular NADase in mammalian tissues, and the characterization of its role on the control of cellular NAD levels indicate that CD38 may serve as a pharmacological target for multiple conditions.

CD38是一种多功能酶,它以烟酰胺腺嘌呤二核苷酸为底物产生第二信使。近年来,CD38也被认为是哺乳动物组织中主要的细胞内 NADases 之一,并且似乎在多种组织和细胞中调节 NAD 的细胞水平。由于 NAD 作为多种信号通路中的关键分子的新兴作用,以及代谢条件,确定调节这种核苷酸合成和降解的细胞机制势在必行。事实上,最近有研究表明 NAD 参与多种生理过程,如胰岛素分泌、能量代谢控制、神经元和心脏细胞存活、支气管收缩、哮喘、衰老和长寿。在哺乳动物组织中发现 CD38作为主要的细胞性 NADase,以及它在控制细胞性 NAD 水平方面的作用的角色塑造表明 CD38可以作为多种条件下的药理学靶点。Keywords: 关键词:CD38, NAD, SIRT1, aging, obesity, metabolic syndrome 38,NAD,SIRT1,衰老,肥胖,代谢症候群

1. INTRODUCTION

1. 引言

Nicotinamide adenine dinucleotide (NAD) is a key cellular metabolite that is involved in cellular energetics. In addition, NAD has recently emerged as a crucial regulator of signaling pathways implicated in multiple physiological conditions [112]. The two main signaling roles of NAD include its importance as a substrate for the generation of second messengers such as cyclic-ADP-ribose (cADPR) [19] and its role as a substrate and regulator of the NAD dependent deacetylases sirtuins [1012]. Both these signaling pathways have been shown to be very important in many physiological conditions from egg fertilization all the way to the cellular mechanisms of aging, longevity, and death [112]. In these regards, a great new interest in NAD functions and metabolism has emerged. In fact, we have seen almost a second discovery of this molecule in recent years [13]. Due to the key role of NAD in cells, it is crucial to characterize the mechanisms that control NAD metabolism. In recent years, we have learned much about the cellular echanisms of NAD synthesis [1214]. Intense research in this field culminated with the discovery of the role of the protein nicotinamide phosphoribosyltransferase (Nampt) (also known as visfatin or PBEF) as a key enzyme involved in de novo synthesis of NAD [15]. In fact, Nampt has been recently shown to modulate NAD levels and some of its cellular functions [15,16]. On the other hand, until recently, very little was known about the mechanisms that regulate NAD degradation in mammalian cells. Our recent studies clearly show that the multifunctional enzyme CD38 is a key enzyme involved in the degradation of NAD and appears to control cellular NAD levels [1719]. In this review, I will focus on the role of CD38 as one of the main cellular NADases, and will discuss its potential role as a pharmacological target for the control of conditions regulated by ellular NAD.

烟酰胺腺嘌呤二核苷酸是细胞内参与细胞能量学的关键代谢产物。此外,NAD 最近已成为一个重要的信号转导途径调节因子涉及多种生理条件[1-12]。NAD 的两个主要信号作用包括作为第二信使产生的底物如环状 adp-ribose (cADPR)[1-9]及其作为 NAD 依赖性去乙酰化酶(deacetylase sirtuins)[10-12]的底物和调节剂的作用。这两个信号通路已经被证明在许多生理条件中非常重要,从卵子受精到衰老、寿命和死亡的细胞机制[1-12]。在这些方面,一个巨大的新兴趣在 NAD 的功能和新陈代谢已经出现。事实上,近年来我们已经发现了几乎第二个这种分子[13]。由于 NAD 在细胞中的关键作用,研究其代谢调控机制至关重要。近年来,我们对 NAD 合成的细胞机制有了很多了解[12-14]。这一领域的紧张研究最终发现了蛋白质烟酰胺磷酸核糖基转移酶(Nampt)(也称为内脂素或 PBEF)的作用,它是参与 NAD 从头合成的关键酶。事实上,Nampt 最近被证明可以调节 NAD 的水平和它的一些细胞功能。另一方面,直到最近,人们对哺乳动物细胞中 NAD 降解的调节机制知之甚少。我们最近的研究清楚地表明,多功能酶 CD38是一个关键酶参与降解 NAD,似乎控制细胞的 NAD 水平[17-19]。在这篇综述中,我将重点介绍 CD38作为一个主要的细胞 NADases 的作用,并将讨论其作为一个潜在的药理作用控制条件由细胞 NAD 调控。

2. CD38 IS A SECOND MESSENGER ENZYME: SYNTHESIS AND DEGRADATION OF CADPR

2. CD38是第二信使酶: CADPR 的合成与降解

2.1. Biochemistry and Metabolism of cADPR

2.1. 钙粘蛋白的生化和代谢

Cyclic-ADP-ribose is a second messenger that induces calcium release from intracellular stores [29]. Cyclic-ADPribose is synthesized from β-NAD+ by an enzymatic activity named ADP-ribosyl cyclase and converted to ADPR in a reaction call cADPR hydrolase (Fig. 1). It is interesting that cADPR metabolism resembles the cyclic AMP system; where a cyclic nucleotide compound with active biological activity (cADPR and cAMP) is hydrolyzed into an inactive non-cyclic compound (ADPR and 5’-AMP). It has to be noted, however, that in many cell types, studied so far, the precise nature of the enzyme(s) responsible for physiological ADP-ribosyl cyclase and cADPR hydrolase activities has not been well established. Nevertheless, ADP-ribosyl cyclase activity has been found across different species spanning from unicellular organisms, invertebrates (sea urchin eggs, Aplysia) to mammalian cells, plants, and parasites suggesting that cADPR metabolism has been preserved in evolution as an ubiquitous second messenger [2039]. ADP-ribosyl cyclases have been found both in soluble and membrane-bound forms. The first characterization of ADP-ribosyl cyclase was performed in Aplysia californica ovotestis [53839]; this soluble 30-KDa enzyme was purified and found to have pure cyclase but no hydrolase activity [39]. It can use β-NAD+ as a substrate, but no α-NAD+ or NADH. It can also metabolize analogs of NAD+, such as nicotinamide guanine dinucleotide (NGD+) and nicotinamide hypoxanthine dinucleotide (NHD+), generating cyclic compounds (cGDPR and cIDPR, respectively) with fluorescent properties, but lacking calcium-releasing activity [40]. These fluorescent compounds are very useful as biochemical tools for studies of ADP-ribosyl cyclase activity [40]. The amino acid sequence of ADP-ribosyl cyclase from Aplysia has considerable homology with the human lymphocyte surface antigen CD38, which led to the discovery that CD38 has also ADP-ribosyl cyclase activity [41]. However, in contrast to Aplysia cyclase, CD38 is a transmembrane protein and has both ADP-ribosyl cyclase and cADPR hydrolase activities. Surprisingly, CD38 bound to the plasma membrane has its catalytic site located on the extracellular domain of the cell, which poses theoretical difficulties for understanding how CD38 generates cADPR in the cytoplasm, where it should be available to interact with calcium channels. Several mechanisms for generation of intracellular cADPR have been proposed, including the translocation of cADPR promoted by CD38 and internalization of CD38 molecules [42], but a clear molecular model remains to be established by further experimental evidence. In cells from vertebrates, the majority of data gathered about cADPR metabolism comes from the studies of cyclase and hydrolase activities of CD38. However, other enzymes though not as extensively characterized as CD38. Another lymphocyte surface antigen named BST-1 (CD157) also has ADP-ribosyl cyclase activity [43]. BST-1 appears to be the product of a gene duplication of CD38 [43].

环二磷酸腺苷核糖是第二信使,诱导钙释放的细胞内商店[2-9]。环状 adpribose 是由 -nad + 合成的 ADP-ribosyl 环化酶,在反应过程中转化为 ADPR 水解酶(图1)。有趣的是,cADPR 代谢类似于环腺苷酸系统,其中一个具有活性生物活性的环核苷酸化合物(cADPR 和 cAMP)被水解成一个非活性的非环化合物(ADPR 和5’-AMP)。然而,需要指出的是,在许多细胞类型中,到目前为止,负责生理性 adp 核糖环化酶和 cADPR 水解酶活性的酶的确切性质还没有得到很好的确定。然而,ADP-ribosyl 环化酶活性已被发现跨越不同物种,包括单细胞生物、无脊椎动物(Aplysia 海胆卵)、哺乳动物细胞、植物和寄生虫,这表明 cADPR 代谢作为一个普遍存在的第二信使在进化过程中被保存了下来[20-39]。Adp 核糖基环化酶存在于可溶性和膜结合形式中。第一角色塑造 adp- 核糖基环化酶是在 Aplysia 进行的[5,38,39] ,这种可溶性的30-KDa 酶被纯化,发现有纯的环化酶,但没有水解酶活性[39]。它可以使用 -nad + 作为底物,但不能使用 -nad + 或 NADH。它还能代谢 NAD + 的类似物,如烟酰胺鸟嘌呤二核苷酸(NGD +)和烟酰胺次黄嘌呤二核苷酸(NHD +) ,生成具有荧光性质的环状化合物(cGDPR 和 cIDPR) ,但缺乏钙释放活性[40]。这些荧光化合物作为研究 adp 核糖环化酶活性的生化工具非常有用[40]。来自 Aplysia 的 adp 核糖环化酶的氨基酸序列与人淋巴细胞表面抗原 CD38具有相当的同源性,从而发现 CD38也具有 adp 核糖环化酶活性[41]。然而,与 Aplysia 环化酶相反,CD38是一个跨膜蛋白,同时具有 adp 核糖环化酶和 cADPR 水解酶活性。令人惊讶的是,结合在质膜上的 CD38的催化位点位于细胞外区域,这给理解 CD38如何在细胞质中产生 cADPR 带来了理论上的困难,在那里它应该可以与钙通道相互作用。细胞内钙粘附分子的产生机制已经被提出,包括 CD38促进的钙粘附分子转位和 CD38分子的内在化,但是一个清晰的分子模型还有待于进一步的实验证实。在脊椎动物细胞中,有关钙粘蛋白代谢的大部分数据来源于对 CD38的环化酶和水解酶活性的研究。然而,其他的酶虽然不像 CD38那样具有广泛的特征。另一种淋巴细胞表面抗原 BST-1(CD157)也具有 adp 核糖环化酶活性[43]。BST-1似乎是 CD38基因重复的产物。Fig. (1) 图(1)

NADase activity: Synthesis and degradation of cyclic ADP-ribose is shown. (A). Synthesis of nicotinic acid adenine dinucleotide phosphate [NAADP+] from nicotinamide adenine dinucleotide phosphate [NADP+] by base-exchange reaction is shown.

NADase 活性: 环状 adp- 核糖的合成和降解。(甲)。以烟酰胺腺嘌呤二核苷酸磷酸为原料,通过碱交换反应合成了烟酸腺嘌呤二核苷酸磷酸酯。

Although it appears that CD38 clearly generates and degrades the second messenger cADPR and plays key roles in the regulation of intracellular calcium transients, CD38 may have other functions. For example, CD38 has been implicated as the enzyme responsible for the generation of other second messengers including NAADP via the base-exchange reaction [4450]. However, the role of CD38 in NAADP generation is still controversial [5051]. In fact, we and others have recently described that CD38 is not necessary for the intracellular generation of NAADP in some mammalian cells [5052]. These indicate that yet another cellular enzyme maybe responsible for some of the functions previously attributed to CD38. It is quite interesting that CD38 appears to be a very inefficient second messenger enzyme, as it will hydrolyze almost a hundred molecules of NAD to generate one molecule of the second messenger cADPR [5354]. In this regard, we have recently focused on the possible role of CD38 not only as a second messenger enzyme, but also as a NADase that can control cellular levels of NAD and its physiological functions [1719].

虽然 CD38可以清楚地产生和降解第二信使 cADPR,并在细胞内钙瞬变的调节中起关键作用,但是 CD38可能还有其他功能。例如,CD38被认为是通过碱基交换反应产生其他第二信使包括 NAADP 的酶[44-50]。然而,CD38在生成 NAADP 中的作用仍然存在争议[50,51]。事实上,我们和其他人最近描述的 CD38是不必要的细胞内生成 NAADP 在一些哺乳动物细胞[50-52]。这些表明,还有另一种细胞酶可能负责一些功能,以前认为是 CD38。有趣的是,CD38似乎是一个非常低效的第二信使酶,因为它将水解近100个 NAD 分子生成一个第二信使 cADPR 分子[53,54]。在这方面,我们最近关注 CD38可能的作用,不仅作为第二信使酶,而且作为一个 NADase,可以控制 NAD 的细胞水平及其生理功能[17-19]。Go to: 去:

3. CD38 IS A CELLULAR NADase: ROLE ON THE DEGRADATION OF NAD AND CONTROL OF NAD LEVELS

3. CD38是一种细胞内的 NADase: 在 NAD 降解和 NAD 水平调控中的作用

CD38 is a multifunctional enzyme, ubiquitously distributed in mammalian tissues [2636]. As discussed above its major enzymatic activity is the hydrolysis of NAD [212426355354]. In fact, as discussed above, CD38 will generate one molecule of cADPR for almost every 100 molecules of NAD hydrolyzed [21]. Until recently, the role of CD38 as a modulator of NAD levels had not been explored. Recently, we postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD levels (Fig. 2). In fact, we examined the NADase activities and NAD levels in a variety of tissues from both wild-type and CD38 deficient mice [17]. In accordance with our hypothesis, we found that tissue levels of NAD in CD38 deficient mice were 10 to 20 fold higher than in wild-type animals [17], a result confirmed by others [55]. In addition, NADase activity was essentially absent in most of the tissues, from CD38 deficient mice [1719]. These data support the novel concept that CD38 is a major regulator of cellular NAD levels. Since CD38 is distributed in nearly every mammalian tissue and cellular compartment, I have postulated that CD38 regulates cellular NAD levels. In particular, CD38 may have a role not only in the regulation of intracellular but also extracellular NAD, and may modulate the availability on extracellular applied NAD in some cellular systems. In addition, the presence of CD38 in different intracellular compartments may have a crucial role on the regulation of NAD functions in specific organelles. The role of nuclear CD38 has been recently explored. CD38 is located at the nuclear membrane and regulates the generation of the second messenger cADPR and nuclear stores calcium release [56]. In addition, we have also found that nuclear CD38 regulates the activity of the nuclear enzymes sirtuins [18]. In particular, we observed that CD38 degrades NAD and decreases the accessibility of NAD to the NAD-dependent acetylase SIRT1 [1819]. It is also possible that generation of nicotinamide by CD38 may regulate SIRT1 activity (Fig. 2) [18]; this is possible by the fact that nicotinamide is an endogenous inhibitor of the SIRT1 enzyme. The potential role of CD38 as a regulator of NAD levels and SIRT1 activity opens the possibility that CD38 may be a regulator of many of the SIRT1 functions including energy homeostasis, obesity, aging, and longevity. Next, I will briefly describe some key aspects of the SIRT1 pathway.

CD38是一种多功能酶,在哺乳动物组织中广泛分布[26-36]。如上所述,它的主要酶活性是 NAD [21,24,26,35,53,54]的水解。事实上,如上所述,CD38将产生一个分子的钙粘蛋白几乎每100分子的 NAD 水解[21]。直到最近,CD38作为 NAD 水平调节剂的作用还没有被探索。最近,我们假设 CD38是哺乳动物细胞中主要的 NADase,并且它调节细胞内 NAD 水平(图2)。实际上,我们检测了野生型和 CD38缺陷小鼠各种组织中 NADase 活性和 NAD 水平。根据我们的假设,我们发现 CD38缺陷小鼠组织中 NAD 的水平比野生型小鼠高10到20倍,这一结果得到了其他小鼠的证实。此外,在 CD38缺陷小鼠的大多数组织中,NADase 活性基本不存在[17-19]。这些数据支持 CD38是细胞 NAD 水平主要调节因子的新概念。由于 CD38分布在几乎所有的哺乳动物组织和细胞区室中,我假设 CD38调节细胞 NAD 水平。特别是 CD38可能不仅参与细胞内 NAD 的调节,而且还参与细胞外 NAD 的调节,并可能调节细胞外 NAD 在某些细胞系统中的有效性。另外,CD38在不同细胞内区域的存在可能对特定细胞器中 NAD 功能的调节起着至关重要的作用。核 CD38的作用最近已经被探索。CD38位于核膜,调节第二信使 cADPR 的产生和核储存钙释放。此外,我们还发现核 CD38调节核酶 sirtuins 的活性[18]。特别是,我们观察到 CD38降解 NAD,降低 NAD 对 NAD- 依赖的乙酰化酶 SIRT1的可接近性。也有可能是 CD38生成烟酰胺可能调节 SIRT1活性(图2)[18] ; 这可能是因为烟酰胺是 SIRT1酶的内源性抑制剂。CD38作为 NAD 水平和 SIRT1活性调节剂的潜在作用开启了 CD38可能是许多 SIRT1功能的调节剂的可能性,包括能量稳态、肥胖、衰老和长寿。接下来,我将简要描述 SIRT1途径的一些关键方面。Fig. (2) 图(2)

Possible mechanism of regulation of SIRT1 and AMPK pathway by CD38 inhibition.

CD38抑制 SIRT1和 AMPK 通路调节机制的研究。

4. SIRT1 AND THE RENEWED INTEREST IN NAD

4. SIRT1和对 NAD 的重新兴趣

4.1. SIRT1, a New Key Regulator Energy Metabolism, Aging and Longevity

4.1. SIRT1,一个新的关键调节能量代谢,老化和长寿

One of the main recent advances on the understanding of energy metabolism, and the subsequent development of metabolic syndrome, has been the discovery and characterization of the metabolic roles of the NAD dependent deacetylases sirtuins. In particular, activation of the sirtuin enzyme SIRT1 has been shown to regulate glucose and fat metabolism and protect animals from high fat diet (HFD)-induced metabolic syndrome, liver steatosis, and obesity. The protective effects of SIRT1 activation maybe mediated by both a systemic melioration of the metabolic syndrome and specific effect on tissue glucose and fat metabolism [5760].

在理解能量代谢以及随后的代谢症候群发展方面的一个主要的最新进展是发现了依赖 NAD 的去乙酰化酶去乙酰化酶在代谢中的作用,并对其进行了角色塑造分析。特别是,sirtuin 酶 SIRT1的激活已被证明可以调节葡萄糖和脂肪代谢,保护动物免受高脂饮食诱导的代谢症候群、肝脏脂肪变性和肥胖。SIRT1激活的保护作用可能是通过代谢症候群的系统改善和对组织葡萄糖和脂肪代谢的特异性作用介导的[57-60]。

4.2. SIRT1 is a Mediator of Caloric Restriction

4.2. SIRT1是卡路里限制的调节器

Several studies have clearly demonstrated that moderate caloric restriction (CR) slows aging, extending life span up to 30–50% [61], furthermore CR can protect animals from the development of metabolic syndrome [61]. Recently it has been shown that the effects of CR are mediated, at least in part, by SIRT1 [62]. SIRT1 uses NAD as a substrate to promote deacetylation of several target proteins. Increased activity of sirtuins leads to life extension in yeast, fruit flies, and C. elegans [6264]. Sirtuins also have an important role in the regulation of body weight, and recently it has been clearly shown that activation of SIRT1 can lead to protection against the development of obesity and liver steatosis [1012].

一些研究已经清楚地表明,适度的热量限制(CR)可以延缓衰老,延长寿命达到30-50% [61] ,此外,CR 可以保护动物免于代谢症候群的发育[61]。最近有研究表明,CR 的影响至少部分是通过 SIRT1[62]介导的。SIRT1以 NAD 为底物促进多种靶蛋白的脱乙酰化反应。去乙酰化酶活性的增加可以延长酵母、果蝇和秀丽线虫的寿命[62-64]。去乙酰化酶在体重调节中也有重要作用,最近已经清楚地表明,激活 SIRT1可以防止肥胖和肝脏脂肪变性的发展[10-12]。

4.3. SIRT1, a Regulator of Systemic and Hepatic Glucose and Fat Metabolism

4.3. SIRT1,全身和肝脏葡萄糖和脂肪代谢的调节剂

SIRT1 regulates energy metabolism, glucose and fat homeostasis both at the systemic and cellular level. In addition to its systemic effects, SIRT1 has liver specific affects on both glucose and fat metabolism [5760]. SIRT1 induced mitochondrial biogenesis, and gene expression in several cell types [1011]. Furthermore, hepatic SIRT1 activation leads to a protection against high fat diet (HFD)-induced steatosis, decreases SREBP1 expression, and the development of inflammation [101159]. In addition, SIRT1 regulates the expression of the antioxidant genes SOD2 (manganese superoxide dismutase), NRF1, and UCP3. In cultured hepatocytes, SIRT1 inhibits glucose induced cellular fat accumulation via an AMPK dependent mechanism [6566]. It appears that all these effects may play a role on the protective effect of SIRT1 activation upon the development of metabolic syndrome, liver steatosis and obesity. Thus, activation of SIRT1 with small molecules, such as resveratrol, may represent a promising strategy for the treatment of metabolic syndrome, and obesity [101119].

SIRT1在全身和细胞水平上调节能量代谢、葡萄糖和脂肪内稳态。除了全身性影响,SIRT1还有肝特异性影响葡萄糖和脂肪代谢[57-60]。SIRT1诱导线粒体生物发生,并在几种细胞类型的基因表达[10,11]。此外,肝脏 SIRT1的激活导致对高脂饮食(HFD)诱导的脂肪变性的保护,降低 SREBP1的表达,以及炎症的发展[10,11,59]。此外,SIRT1还调节抗氧化剂基因 SOD2(超氧化物歧化酶)、 NRF1和 UCP3的表达。在培养的肝细胞中,SIRT1通过 AMPK 依赖机制抑制葡萄糖诱导的细胞脂肪堆积[65,66]。似乎所有这些作用都可能在 SIRT1激活对代谢症候群、肝脏脂肪变性和肥胖的发展起到保护作用。因此,利用小分子激活 SIRT1,如白藜芦醇,可能是治疗代谢症候群和肥胖症的一种有前途的策略。

4.4. NAD, Sirtuins (SIRT1) and Obesity

4.4. NAD、 Sirtuins (SIRT1)与肥胖

NAD and nicotinamide play key roles in many cellular functions [11119]. In addition to its well known importance in energy metabolism, NAD and nicotinamide also play a role in signal transduction. New evidence suggests that NAD and nicotinamide are regulators of the NAD dependent deacetylases (sirtuins also known as SIRT enzymes), which modulates, obesity, energy metabolism, aging, and longevity [10125766]. In fact, the drug resveratrol, an activator of the sirtuin enzymes, has been recently shown to protect animals against high fat (caloric) diet (HFD)-induced obesity, via an increase in energy expenditure [101119]. In addition, activation of SIRT1 enzymes in mice feed HFD ameliorate pathological effects of obesity (including glucose tolerance), and increase longevity [10]. These data indicates that SIRT1 enzyme not only prevent obesity, but also promote salutary health benefits in HFD feed animals [101119]. The effects of SIRT1 on obesity and energy metabolism are, at least in parts, mediated by deacetylation and activation of peroxisome proliferato ractivated receptor α coactivator, PGC1α [101119] (Fig. 3). Very strong evidence supports the notion that PGC1α is a key regulator of energy metabolism and mitochondrial biogenesis [67]. However, to date, the intracellular mechanisms that regulate SIRT1 mediated activation of PGC1α, inmammalian cells, have not been elucidate.

NAD 和烟酰胺在许多细胞功能中起关键作用[1-11,19]。除了它在能量代谢方面的重要性,NAD 和烟酰胺也在信号转导中发挥作用。新的证据表明 NAD 和烟酰胺是 NAD 依赖性去乙酰化酶(sirtuins 也被称为 SIRT 酶)的调节剂,该酶能调节肥胖、能量代谢、衰老和寿命[10-12,57-66]。事实上,药物白藜芦醇,一种去乙酰化酶的激活剂,最近被证明可以通过增加能量消耗来保护动物免受高脂肪(卡路里)饮食(HFD)引起的肥胖。此外,在小鼠饲料中激活 SIRT1酶改善肥胖的病理效应(包括葡萄糖耐量) ,并延长寿命[10]。这些数据表明,SIRT1酶不仅可以防止肥胖,而且可以促进 HFD 饲料动物的健康益处[10,11,19]。SIRT1对肥胖和能量代谢的影响,至少部分是通过过氧化物酶体增殖物激活受体辅激活剂 pgc1的去乙酰化和激活介导的(图3)。非常有力的证据支持 pgc1是能量代谢和线粒体生物发生的关键调节因子的观点[67]。然而,迄今为止,细胞内调节 SIRT1介导的 pgc1的激活机制,在哺乳动物细胞,尚未阐明。Fig. (3) 图(3)

Possible mechanism of regulation of SIRT1, PGC1 pathway, and obesity by CD38 inhibition.

CD38抑制剂调节 SIRT1、 PGC1通路与肥胖的可能机制。

4.5. SIRT1 has Multiple Cellular Targets

4.5. SIRT1具有多个细胞靶点

In addition to the deacetylation of PGC-1 [112], SIRT1deacetylates other diverse substrates including p53, forkheadtranscription factor (FOXO), NF-κβ, Ku70, MyoD, LXR ,and histones. Thus, it influences gene silencing, apoptosis, stress resistance, cellular senescence, fat and glucose metabolism. The combination of these cellular functions might contribute to the physiological effects of SIRT1. Despite the extensive studies of SIRT1 function, the regulation of SIRT1 is poorly understood.

除了 PGC-1[1-12]的去乙酰化作用外,sirt1deacetylate 还可以使 p53、叉头转录因子(FOXO)、 nf- 、 Ku70、 MyoD、 LXR 和组蛋白等多种底物发生去乙酰化作用。因此,它影响基因沉默、细胞凋亡、抗应激、细胞衰老、脂肪和葡萄糖代谢。这些细胞功能的结合可能有助于 SIRT1的生理效应。尽管对 SIRT1的功能进行了广泛的研究,但是对 SIRT1的调控机制还知之甚少。Go to: 去:

5. CD38 AS A REGULATOR OF NAD AND SIRT1

5. CD38作为 NAD 和 SIRT1的调节因子

5.1. CD38 and NAD Metabolism

5.1. CD38与 NAD 代谢

As discussed above, CD38 is an enzyme that has been implicated on the generation of the second messenger cyclic- ADP-ribose (cADPR) [1226]. However, its main enzymatic activity is the hydrolysis of NAD to nicotinamide and ADPR. We postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD and nicotinamide levels. In fact, our recent publications indicate that in CD38 deficient mice, tissue levels of NAD are several folds higher than in wild type animals [17]. In addition, we observed that NADase activity is essentially absent in several tissues from CD38 deficient mice [17].

如上所述,CD38是一种与第二信使环二磷酸核糖(cADPR)[12-26]的产生有关的酶。但其主要的酶活性是 NAD 水解生成烟酰胺和 ADPR。我们假设 CD38是哺乳动物细胞中主要的 NADase,它调节细胞内 NAD 和烟酰胺水平。事实上,我们最近的出版物表明,在 CD38缺陷小鼠中,NAD 的组织水平比野生型动物高几倍[17]。此外,我们观察到,在 CD38缺陷小鼠的一些组织中,NADase 活性基本上不存在。

5.2. CD38, a Regulator of SIRT1 Activity?

5.2. CD38,SIRT1活性的调节因子?

We proposed that by modulating availability of NAD and nicotinamide to the SIRT1 enzyme, CD38 regulates SIRT1 activity. These findings have strong implications for the understanding of the basic mechanisms that modulate obesity, metabolic syndrome, energy homeostasis, longevity, and aging.

我们提出,通过调节 NAD 和烟酰胺对 SIRT1酶的有效性,CD38可以调节 SIRT1的活性。这些发现对于理解调节肥胖、代谢症候群、能量平衡、长寿和衰老的基本机制有着重要的意义。

5.3. CD38 and Obesity

5.3. CD38与肥胖

A correlation has been observed between chromosome 4 near marker D4S403, where the CD38 gene is located, and the development of metabolic syndrome that refers to the clustering of disease conditions such as obesity, insulin resistance, hyperinsulinemia, and dyslipidemia [68]. However, to date, except for our studies [1719], no other studies have been published on the role of CD38 on diet-induced obesity. CD38 regulates SIRT1 via a Non-cADPR mediated mechanism. Although CD38 has been implicated as the enzyme responsible for the generation of the second messenger cADPR [5], CD38 also appears to have cADPR-independent functions. In the case of regulation of NAD, SIRT1 activity, and obesity, our data indicates that CD38 appears to do it via a cADPR independent way, but a SIRT1 dependent mechanism [19]. The regulation of NAD by CD38 and its implication for pharmacological approaches aim at increasing SIRT1 activity. We proposed that by augmenting NAD and decreasing nicotinamide levels, inhibition of CD38 will not only increase SIRT1 activity but also will increase the sensitivity of SIRT1 to its pharmacological agonists such as resveratrol. In fact, we have previously observed that the activation of recombinant SIRT1 by resveratrol is inhibited by the addition of active recombinant CD38 to the reaction media [17].

在 CD38基因所在的 D4S403标记附近的4号染色体与代谢症候群的发展之间已经观察到了一种相关性,这种相关性指的是肥胖、胰岛素抵抗、高胰岛素血症和血脂异常等疾病状况的聚集。然而,到目前为止,除了我们的研究[17-19] ,没有其他研究发表关于 CD38在饮食诱导肥胖中的作用。CD38通过非钙粘蛋白介导的机制调节 SIRT1。虽然 CD38被认为是负责产生第二信使 cADPR 的酶[5] ,但是 CD38似乎也具有与 cADPR 无关的功能。对于 NAD、 SIRT1活性和肥胖的调节,我们的数据表明 CD38似乎是通过一种独立于 cADPR 的方式,而是一种 SIRT1依赖机制[19]。CD38对 NAD 的调节作用及其对药理学研究的意义旨在提高 SIRT1的活性。我们提出,通过增加 NAD 和降低烟酰胺水平,抑制 CD38不仅可以提高 SIRT1的活性,而且可以提高 SIRT1对白藜芦醇等药理激动剂的敏感性。事实上,我们之前已经观察到白藜芦醇对重组 SIRT1的激活被活性重组 CD38添加到反应介质[17]所抑制。

6. CD38 INHIBITORS

6. CD38抑制剂

To date a few CD38 inhibitors have been reported including NAD analogs (arabiono-NAD), nicotinamide derivatives (nicotinamide and nicotinic acid), reducing agents (such as dithiothreitol), and other unrelated compounds (Fig. 4; and reference [2771]). The compound 2,2′-dihydroxyazobenzene (DAB) has been recently shown to protect against cardiac dysfunction-induced by angiotensin II [69]. At the present time, the search for specific and potent CD38 inhibitors remains elusive. However, a recent report on a new CD38 assays indicates that an intensive search for CD38 inhibitors is going at this time [70], and it maybe a question of time before potent and specific CD38 inhibitors are available atleast for research. In any case, some important aspects of the search for CD38 inhibitors deserve further discussion. First, it is important to say that it is possible that molecules that inhibit CD38 may also inhibit SIRT1 [71]. In fact, SIRT1 and CD38 have several similarities in their enzymatic and catalytical properties [71], and CD38 has been proposed as a model enzyme for the study of the mechanism of SIRT1 catalysis [71]. Both SIRT1 and CD38 degrades NAD to nicotinamide and an ADPR derivative. Furthermore, both enzymes are capable of base-exchange reaction (Fig. 1B). In this regard, inhibitors of CD38 may also have effects upon SIRT1 activity, a potential undesirable “side-effect”. Secondly, as discussed above CD38 have other functions that are mediated by the generation of calcium regulating second messengers such as smooth muscle contraction, cell death, and apoptosis, neural and hormonal signaling, egg fertilization and others [2921697273]. In this regard, CD38 inhibitors may have beneficial effects upon conditions, where cellular calcium homeostasis is deregulated such as in hypertension, cardiac ischemia, asthma and dysfunctional labor [2921697273]. On the other hand, CD38 has been implicated in the secretion and function of hormones such as oxytocin and ACTH [7475], and may modulate maternal and social behavior [75]. These roles indicate that inhibition of CD38 may have potential deleterious effects. Potential immunologic dysfunction may be one of the worst possible “side-effects” of CD38 inhibitors. It has been shown that CD38 plays a key role in the mechanism by which the organism fights bacterial infection [76], and knockout of CD38 leads to increase susceptibility to lethal bacterial infection [76]. Despite these limitations, the search for CD38 inhibitors and the determination of their potential therapeutic roles will generate key new data that will provide new insights on multiple physiological and pathological conditions, and CD38 inhibitors may hold the key to new therapeutic strategies to multiple metabolic and inflammatory conditions.

迄今为止,已报道了一些 CD38抑制剂,包括 NAD 类似物(arabiono-NAD)、烟酰胺衍生物(烟酰胺和烟酸)、还原剂(如二硫苏糖醇)和其他无关化合物(图4; 和参考文献[27,71])。化合物2,2’-二羟基偶氮苯(DAB)最近被证明可以防止血管紧张素 II (69)引起的心脏功能障碍。目前,寻找特异的、有效的 CD38抑制剂仍然是一个难题。然而,最近一项关于新的 CD38分子检测的报告表明,对 CD38抑制剂的深入研究正在进行[70] ,而且至少在研究上有效和特异的 CD38抑制剂可用之前,可能还有一个时间问题。无论如何,CD38抑制剂研究的一些重要方面值得进一步讨论。首先,重要的是说抑制 CD38的分子也可能抑制 SIRT1[71]。实际上,SIRT1和 CD38在酶和催化性质上有许多相似之处[71] ,CD38已被提出作为研究 SIRT1催化机理的模型酶[71]。SIRT1和 CD38都能将 NAD 降解为烟酰胺和 ADPR 衍生物。此外,这两种酶都能进行碱基交换反应(图1B)。在这方面,CD38抑制剂也可能对 SIRT1活性有影响,一个潜在的不良“副作用”。其次,正如上面所讨论的,CD38还有其他的功能,这些功能是由钙调节的第二信使的产生介导的,如平滑肌肉收缩,细胞死亡,细胞凋亡,神经和激素信号传导,卵子受精等等。在这方面,CD38抑制剂可能对细胞钙稳态解除的条件有益,如在高血压,心肌缺血,哮喘和功能失调的分娩[2-9,21,69,72,73]。另一方面,CD38与催产素和促肾上腺皮质激素(ACTH)等激素的分泌和功能有关,并可能调节母性和社会行为。这些作用表明,抑制 CD38可能具有潜在的有害影响。潜在的免疫功能障碍可能是 CD38抑制剂最严重的“副作用”之一。研究表明,CD38在机体对抗细菌感染的机制中起着关键作用,而 CD38基因的敲除会增加致死性细菌感染的易感性。尽管存在这些局限性,对 CD38抑制剂的研究及其潜在治疗作用的确定将产生关键的新数据,这些数据将为多种生理和病理状况提供新的见解,而 CD38抑制剂可能是多代谢和炎症状况的新治疗策略的关键。Fig. (4) 图(4)

A typical CD38-NADase assay using etheno-NAD and recombinant CD38. Activity is inhibited by the reaction product nicotinamide. NADase acitivity is defined using etheno-NAD as a substrate and adding CD38 in the presence or absence of 1mM nicotinamide. Samples were incubated for 10 min.

利用乙烯基 nad 和重组 CD38建立典型的 CD38-NADase 检测方法。反应产物烟酰胺抑制活性。以乙烯基 -nad 为底物,在有或无烟酰胺存在的条件下加入 CD38,定义了 NADase 活性。样品培养10分钟。

7. CONCLUSION

7. 结论

Finally, it is important to discuss the fact that NADases are present in many other organisms such as bacteria and protozoans [7778]. Group A streptococci produce several exoproteins that are thought to contribute to the pathogenesis of human infection. One of these proteins is a NAD+-glycohydrolase (NADase). When group A streptococci are bound to the surface of epithelial cells in vitro, pores in the cell membrane are form and bacterial NADase is delivered to the epithelial cell cytoplasm. In vitro, intoxication of keratinocytes with NADase is associated with cytotoxic effects and induction of apoptosis [77]. In this regard, bacterial NADase plays a key role on the virulence of some bacteria [77]. Furthermore, we have recently described that the parasite Toxoplasma gondii has a NADase/ADP-ribosyl cyclase [7879]. Furthermore, in toxoplasma generation of cADPR, induced by abscisic acid, plays a key role in the mechanisms of cell invasion, differentiation and egress [7880]. Inhibition of the NADase/ADP-ribosyl cyclase may be a novel target for pharmacological therapy against parasitic infection [7880]. It is possible that CD38 inhibitors may cross many species barriers and may also be effective against microorganismal NADases, and maybe use for the treatment of microbiotic infection. In any case, search for specific bacterial and protozoa NADase inhibitors may also be of great importance. The recent development on the understanding of the catalytic properties of CD38 and the development of assays to study its NADase and base-exchange reaction maybe the initial step for the development of CD38 and species specific NADase inhibitors that may have multiple potential therapeutic roles in many human diseases. The future in this field is extremely exciting and provides promises of new and exciting pharmacological tools, let the hunt begin.

最后,重要的是要讨论这样一个事实,即 NADases 存在于许多其他生物体中,如细菌和原生动物[77,78]。A 组链球菌产生的几种外泌蛋白被认为有助于人类感染的发病机制。其中一种蛋白质是 NAD +-糖水解酶(NADase)。当 a 组链球菌与上皮细胞表面结合时,细胞膜上形成孔隙,细菌 NADase 进入上皮细胞胞浆。在体外,角质形成细胞中 NADase 中毒与细胞毒作用和诱导凋亡有关[77]。在这方面,细菌 NADase 在一些细菌的毒力中起着关键作用[77]。此外,我们最近描述的寄生虫弓形虫有一个 nadase/adp 核糖环化酶[78,79]。此外,在由脱落酸诱导的弓形虫钙粘蛋白的产生过程中,钙粘蛋白在细胞侵袭、分化和迁出的机制中起着关键作用。抑制 nadase/adp 核糖环化酶可能是抗寄生虫感染药物治疗的新靶点[78-80]。CD38抑制剂可能跨越多种物种屏障,也可能对微生物 NADases 有效,可能用于治疗微生物感染。在任何情况下,寻找特定的细菌和原生动物 NADase 抑制剂可能也是非常重要的。近年来对 CD38催化性质的研究进展,以及对其 NADase 和碱基交换反应的研究进展,可能是开发 CD38和具有多种潜在治疗作用的物种特异性 NADase 抑制剂的起始步骤。在这个领域的未来是非常令人兴奋的,并提供了新的和令人兴奋的药理学工具的承诺,让搜寻开始。

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