As KTWGQYWQV, ITDQVPFSV is a gp100 epitope that is recognized in the context of HLA-A2 by tumor-infiltrating lymphocytes (TIL) in vivo. PMEL 17 could be a melanogenic enzyme.

Definition

Intermedin (IMD) is a novel member of the calcitonin gene-related peptide (CGRP) family which also includes adrenomedullin (AM).

Discovery

In early 2004, a novel peptide was independently identified by two separate groups. Roh and colleagues initially identified human IMD from an expressed sequence tag and used phylogenetic profiling to identify the peptide in other mammals and nonvertebrates. Immunohistochemical analysis of peptide expression revealed that high levels of the peptide and/or preprohormone were present in cells within the intermediate lobe of the pituitary; hence, the group called the peptide intermedin1. At the same time, Takei et al. were examining a rat cDNA library for potential mammalian homologs of the five identified puffer fish adrenomedullin (AM) family members. They identified a 148-amino acid preprohormone that was processed into a 47-amino acid, mature peptide with 33% sequence homology to AM and 71% homology to puffer fish AM2 and thus referred to the peptide as mammalian AM22.

Structural Characteristics

The human IMD gene is believed to encode a prepropeptide of 148 amino acids with a signal peptide for secretion at the N terminus, which may generate a 47-amino acid mature peptide (IMD1–47) i.e. IMDL and a shorter 40-amino acid one (IMD8–47) i.e.  IMDS by proteolytic cleavage at the N-terminal proximate basic residues followed by an amidated C terminus. Intermedins has a stretch of 47 residues at the C terminus is flanked by dibasic proteolytic cleavage sites at the N terminus and an a-amidation donor residue at the C terminus. The putative mature region of intermedin shares 28% sequence identity with ADM and 20% with CGRP. Importantly, Intermedins adopts an N-terminal disulfide- bonded loop leading into an a-helix, followed by a disordered structure that is shared by all calcitonin/CGRP family peptides. Analysis of orthologous intermedins indicate that the positions of N-terminal dibasic cleavage sites vary by a few amino acids among different species, whereas an arginine residue seven amino acids downstream of the dibasic cleavage motif of human intermedin is conserved in all species, suggesting that the mature intermedin from human and other species could be a 40-amino acid peptide3.

Mode of action

Intermedin activates the cAMP-dependent Pathway via the CGRP Receptor— A study shows that treatment of human neuroblastoma SK-N-MC cells and rat L6 skeletal myoblast cells, known to express different levels of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMPs), with synthetic intermedin peptides resulted in increased cAMP production, whereas  treatment with the  truncated amidated intermedin fragment (intermedin (IMD)- (17–47)), or a 31-amino acid peptide from the preproregion of human intermedin (prointermedin-(55–85)) had no effect in either cell line. This implied that a-amidation and residues 8–16 of intermedin are important for intermedin bioactivity. Further it was also shown that the stimulatory effect of intermedin was suppressed by cotreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP-(8–37)) in L6 cells3.

Functions

Intermedin/Adrenomedullin-2 elevate blood pressure and inhibit food and water intake - Intermedin is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. It has been reported that administration of IMD into the lateral cerebroventricle (i.c.v.) of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by i.c.v. administration of phentolamine indicating that IMD activates the sympathetic nervous system. I.c.v administration of IMD also inhibited food and water intake. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. A study indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both the AM and CGRP receptors4.

Expression of the Counter-Regulatory Peptide Intermedin is Augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes - A study was done using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY), to examine the presence of myocardial oxidative stress, concentric hypertrophy expression of IMD, AM and receptor components. It was found that in left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%) and IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered and abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. This implied that robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension5.

References

1.     Roh J, Chang CL, Bhalla A, Klein C, and Hsu SYT (2004). Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complex. J. Biol. Chem., 279, 7264–7274.

2.     Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, and Miyano S (2004). Novel fish-derived adrenomedullin in mammals: structure and possible function. Peptides, 25, 1643- 1656.

3.     Jaesook Roh, Chia Lin Chang, Alka Bhalla, Cynthia Klein, and Sheau Yu Teddy Hsu (2004). Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family Peptide Acting through the Calcitonin Receptor-liken Receptor/Receptor Activity-modifying Protein Receptor Complexes. J. Bio. Chem., 279 (20), 7264–7274.

4.     Taylor MM, Bagley SL, Samson WK (2004). Intermedin/Adrenomedullin-2 acts within the central nervous system to elevate blood pressure and inhibit food and water intake. Am. J Physiol. Regul. Integr. Comp. Physiol., 288, R919-R927.

5.     David Bell, Y Zhao, Francis PG. McCoy, Adrian Devine and Barbara J. McDermott (2008). Expression of the Counter-Regulatory Peptide Intermedin is augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes. Cell. Physiol. Biochem., 21, 409-420.

化學預防肽是有助于預防疾?。ɡ绨┌Y或糖尿病）的發(fā)作或發(fā)展的肽。這些肽可以源自天然來源，例如大豆或牛奶，也可以來自肽模擬物的設計，也可以源自使用合成肽進行的肽篩選。據(jù)認為，這些肽中的某些可以充當細胞周期的調(diào)節(jié)劑，其調(diào)節(jié)使細胞通過復制周期前進所需的蛋白質(zhì)的產(chǎn)生和功能。另外，現(xiàn)在有越來越多的證據(jù)表明特定的飲食模式，食物和飲料以及其他飲食物質(zhì)可以而且確實可以預防癌癥。越來越多的流行病學研究表明，食物，營養(yǎng)和身體活動在預防和改變癌癥過程中很重要。包括植物蛋白酶抑制劑，乳鐵蛋白，乳鐵蛋白，凝集素和lunasin在內(nèi)的不同類型的食物蛋白和多肽似乎起著化學預防劑的作用。如今，蛋白質(zhì)和多肽被認為是一組營養(yǎng)保健品，在預防癌癥的不同階段（包括起始，促進和進展）方面顯示出潛力。此外，已經(jīng)發(fā)現(xiàn)在植物中發(fā)現(xiàn)的一些蛋白酶抑制劑，例如豆類和大豆，是有效的癌發(fā)生抑制劑。致癌作用是引發(fā)和促進癌癥的過程。 Bowman-Birk抑制劑和Kunitz胰蛋白酶抑制劑就在其中。目前，這些化合物在致癌作用中的生物學功能主要歸因于抑制癌細胞的侵襲和轉(zhuǎn)移，但是，其作用機理仍不完全清楚，需要進一步研究以充分闡明它們。

多肽H2N-Ile-Thr-Asp-Gln-Val-Pro-Phe-Ser-Val-COOH的合成步驟：

1、合成CTC樹脂：稱取1.76g CTC Resin（如初始取代度約為1.04mmol/g）和2.2mmol Fmoc-Val-OH于反應器中，加入適量DCM溶解氨基酸（需要注意，此時CTC樹脂體積會增大好幾倍，避免DCM溶液過少），再加入5.49mmol DIPEA（Mw：129.1,d：0.740g/ml），反應2-3小時后，可不抽濾溶液，直接加入1ml的HPLC級甲醇，封端半小時。依次用DMF洗滌2次，甲醇洗滌1次，DCM洗滌一次，甲醇洗滌一次，DCM洗滌一次，DMF洗滌2次（這里使用甲醇和DCM交替洗滌，是為了更好地去除其他溶質(zhì)，有利于后續(xù)反應）。得到  Fmoc-Val-CTC Resin。結構圖如下：

2、脫Fmoc：加3倍樹脂體積的20%Pip/DMF溶液，鼓氮氣30分鐘，然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Val-CTC Resin 。（此步驟脫除Fmoc基團，茚三酮檢測為藍色，Pip為哌啶）。結構圖如下：

3、縮合：取5.49mmol Fmoc-Ser(tBu)-OH 氨基酸，加入到上述樹脂里，加適當DMF溶解氨基酸，再依次加入10.98mmol DIPEA，5.22mmol HBTU。反應30分鐘后，取小樣洗滌，茚三酮檢測為無色。用2倍樹脂體積的DMF 洗滌3次樹脂。（洗滌樹脂，去掉殘留溶劑，為下一步反應做準備）。得到Fmoc-Ser(tBu)-Val-CTC Resin。氨基酸：DIPEA：HBTU：樹脂=3：6：2.85：1（摩爾比）。結構圖如下：

4、依次循環(huán)步驟二、步驟三，依次得到

H2N-Ser(tBu)-Val-CTC Resin

Fmoc-Phe-Ser(tBu)-Val-CTC Resin

H2N-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Pro-Phe-Ser(tBu)-Val-CTC Resin

H2N-Pro-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

H2N-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

H2N-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

H2N-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Thr(tBu)-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

H2N-Thr(tBu)-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

Fmoc-Ile-Thr(tBu)-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin

以上中間結構，均可在專肽生物多肽計算器-多肽結構計算器中，一鍵畫出。

最后再經(jīng)過步驟二得到 H2N-Ile-Thr(tBu)-Asp(OtBu)-Gln(Trt)-Val-Pro-Phe-Ser(tBu)-Val-CTC Resin，結構如下：

5、切割：6倍樹脂體積的切割液（或每1g樹脂加8ml左右的切割液），搖床搖晃 2小時，過濾掉樹脂，用冰無水乙醚沉淀濾液，并用冰無水乙醚洗滌沉淀物3次，最后將沉淀物放真空干燥釜中，常溫干燥24小試，得到粗品H2N-Ile-Thr-Asp-Gln-Val-Pro-Phe-Ser-Val-COOH。結構圖見產(chǎn)品結構圖。

切割液選擇：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前兩種適合沒有容易氧化的氨基酸，例如Trp、Cys、Met。第三種適合幾乎所有的序列。）

6、純化凍干：使用液相色譜純化，收集目標峰液體，進行凍干，獲得蓬松的粉末狀固體多肽。不過這時要取小樣復測下純度 是否目標純度。

7、最后總結：

杭州專肽生物技術有限公司（ALLPEPTIDE http://tsjxdd.com）主營定制多肽合成業(yè)務，提供各類長肽，短肽，環(huán)肽，提供各類修飾肽，如：熒光標記修飾（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基團修飾肽（疊氮、炔基、DBCO、DOTA、NOTA等），同位素標記肽（N15、C13），訂書肽（Stapled Peptide）,脂肪酸修飾肽（Pal、Myr、Ste），磷酸化修飾肽（P-Ser、P-Thr、P-Tyr），環(huán)肽（酰胺鍵環(huán)肽、一對或者多對二硫鍵環(huán)），生物素標記肽，PEG修飾肽，甲基化修飾肽

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