CART peptides, especially CART (55-102) appear to have an important function in the regulation of energy homeostasis. Intracerebroventricular administration of CART (55-102) reduces appetite and stimulates energy expenditure, whereas injection of the peptide into specifichypothalamic nuclei increases food intake.

二硫鍵廣泛存在與蛋白結(jié)構(gòu)中，對(duì)穩(wěn)定蛋白結(jié)構(gòu)具有非常重要的意義，二硫鍵一般是通過(guò)序列中的2個(gè)Cys的巰基，經(jīng)氧化形成。
 

形成二硫鍵的方法很多：空氣氧化法，DMSO氧化法，過(guò)氧化氫氧化法等。
 

二硫鍵的合成過(guò)程，  可以通過(guò)Ellman檢測(cè)以及HPLC檢測(cè)方法對(duì)其反應(yīng)進(jìn)程進(jìn)行監(jiān)測(cè)。  
   

如果多肽中只含有1對(duì)Cys，那二硫鍵的形成是簡(jiǎn)單的。多肽經(jīng)固相或液相合成，然后在pH8-9的溶液中進(jìn)行氧化。      
 

當(dāng)需要形成2對(duì)或2對(duì)以上的二硫鍵時(shí)，合成過(guò)程則相對(duì)復(fù)雜。盡管二硫鍵的形成通常是在合成方案的最后階段完成，但有時(shí)引入預(yù)先形成的二硫化物是有利于連合或延長(zhǎng)肽鏈的。通常采用的巰基保護(hù)基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團(tuán)。我們分別列出兩種以2-Cl樹(shù)脂和Rink樹(shù)脂為載體合成的多肽上多對(duì)二硫鍵形成路線：
 

二硫鍵反應(yīng)條件選擇    
 

 二硫鍵即為蛋白質(zhì)或多肽分子中兩個(gè)不同位點(diǎn)Cys的巰基（-SH）被氧化形成的S-S共價(jià)鍵。 一條肽鏈上不同位置的氨基酸之間形成的二硫鍵，可以將肽鏈折疊成特定的空間結(jié)構(gòu)。多肽分 子通常分子量較大，空間結(jié)構(gòu)復(fù)雜，結(jié)構(gòu)中形成二硫鍵時(shí)要求兩個(gè)半胱氨酸在空間距離上接近。 此外，多肽結(jié)構(gòu)中還原態(tài)的巰基化學(xué)性質(zhì)活潑，容易發(fā)生其他的副反應(yīng)，而且肽鏈上其他側(cè)鏈 也可能會(huì)發(fā)生一系列修飾，因此，肽鏈進(jìn)行修飾所選取的氧化劑和氧化條件是反應(yīng)的關(guān)鍵因素， 反應(yīng)機(jī)理也比較復(fù)雜，既可能是自由基反應(yīng)，也可能是離子反應(yīng)。      

反應(yīng)條件有多種選擇，比如空氣氧化，DMSO氧化等溫和的氧化過(guò)程，也可以采用H2O2，I2， 汞鹽等激烈的反應(yīng)條件。
 

空氣氧化法： 空氣氧化法形成二硫鍵是多肽合成中最經(jīng)典的方法，通常是將巰基處于還原態(tài)的多肽溶于水中，在近中性或弱堿性條件下（PH值6.5-10），反應(yīng)24小時(shí)以上。為了降低分子之間二硫鍵形成的可能，該方法通常需要在低濃度條件下進(jìn)行。
 

碘氧化法：將多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘進(jìn)行氧化，反應(yīng)15-40min。當(dāng)肽鏈中含有對(duì)碘比較敏感的Tyr、Trp、Met和His的殘基時(shí)，氧化條件要控制的更精確，氧化完后，立即加入維生素C或硫代硫酸鈉除去過(guò)量的碘。 當(dāng)序列中有兩對(duì)或多對(duì)二硫鍵需要成環(huán)時(shí)，通常有兩種情況：
 

自然隨機(jī)成環(huán):       序列中的Cys之間隨機(jī)成環(huán)，與一對(duì)二硫鍵成環(huán)條件相似；
 

定點(diǎn)成環(huán)：       定點(diǎn)成環(huán)即序列中的Cys按照設(shè)計(jì)要求形成二硫鍵，反應(yīng)過(guò)程相對(duì)復(fù)雜。在 固相合成多肽之前，需要提前設(shè)計(jì)幾對(duì)二硫鍵形成的順序和方法路線，選擇不同的側(cè)鏈 巰基保護(hù)基，利用其性質(zhì)差異，分步氧化形成兩對(duì)或多對(duì)二硫鍵。       通常采用的巰基保護(hù) 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團(tuán)。

Definition

Cocaine- and amphetamine- regulated transcript (CART) peptides, derived from proCART polypeptide in humans are neuropeptides expressed in the brain1. They mainly serve as inhibitors of food intake1.

Discovery

CART was first purified by gel filtration and sequenced in 1980 from rat hypothalamus2.

Classification

CART is a neuropeptide family member2.

Structural Characteristics

CART gene encodes a peptide of either 129 or 116 amino acid residues in rat whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a precursor of 102 or 89 residues3. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule.  Various CART peptides can be generated from the precursor: CARTs (55-102), (85-102), (55-76) and (62-76)3.  CART (55-102) contains three disulphide bridges that are required for its function4.                                                                    

Mode of action

The mode of action of CART peptides has not been fully established. Several studies have observed that they may function through CART receptors in turn activating down stream signaling pathways5.

Functions

CART is distributed in the central nervous system and periphery and has many physiological roles.  It elicits similar behavior as cocaine.  CART has been shown to have variety of effects on dopamine6.  It increases blood pressure, has variety of behavioral effects and influences nociception6.  In the hypothalamus CART mainly regulates energy homeostasis6.  CART is also an endogenous inhibitor of food intake6.  CART peptides are also important in anxiety, pain, arousal, startle response, regulation of calcium channels, and neuroendocrine hormone secretion5.

References

1.     Thim L, Kristensen P, Larsen PJ, Wulff BS (1998). CART, a new anorectic peptide. Int J Biochem Cell Biol, 30(12), 1281-4.

2.     Spiess J and Vale W (1980). Multiple forms of somatostatin-like activity in rat hypothalamus. Biochemistry 19, 2861–66.

3.     Dylag T, Kotlinska J, Rafalski P, Pachuta A, Silberring J (2006). The activity of CART peptide fragments. Peptides, 27(8), 1926-33.

4.     Murphy KG (2005). Dissecting the role of cocaine and amphetamine-regulated transcript (CART) in the control of appetite. Briefing in Functional Genomics and proteomic, 4 (2),  95–111.

5.     Rogge G, Jones D, Hubert GW, Lin Y & Kuhar MJ (2008). CART peptides: regulators of body weight, reward and other functions. Nature Reviews Neuroscience, 9, 747-758.

6.      Vicentic A, Jones DC (2007). The CART (cocaine- and amphetamine-regulated transcript) system in appetite and drug addiction. J Pharmacol Exp Ther., 320(2), 499-506.