Monday, 15 July 2019

Benefits of Peptides

Benefits of Peptides

Benefits of Peptides

A peptides is essentially comprised of  L -amino acid residues, and the peptide backbone may be critical to provide physicochemical and functional properties. Diverse functionalities induced by peptides in natural protein hydrolysates are being received with much interest in food industries or alternative-medicinal food sciences. The latest year’s researches have provided an interesting physiological potential that several smaller di- and tripeptides elicit not only anti-hypertension effect 1 via an angiotensin I-converting enzyme (ACE) inhibition, 2 but also Ca 2+ channel blocking activity, 3,4 vasorelaxation action 5,6 and anti-atherosclerotic effect. 7 Hence, we can prospect a hopeful future of small peptides based on this review, in which the findings from a series of studies conducted in the past decades are summarized.

Benefits of Peptides


  • Regulation of Renin–Angiotensin System by Peptides


Inhibition of Renin–Angiotensin System


Antihypertensive FOSHU products are accepted as a food that possesses ACE inhibitory action. So far, 120 antihypertensive or ACE inhibitory items have been developed from natural food materials such as sardine muscle, milk, dried bonito, sesame, seaweed and royal jelly. ACE, a zinc-containing carboxydipeptidase, mainly located in the lung, kidney and vessel, can convert inactive circulating decapeptide angiotensin (Ang) I to active octapeptide Ang II at the pressor metabolic system of the renin–angiotensin (RAS). 8 Ang II stimulates aldosterone release from the adrenal zona glomerulosa and salt retention in the renal proximal tubules. In vessels, once Ang II binds to Ang II type-1 receptor (AT 1 -R), it activates a series of G-protein–related signaling cascades. Ang II is also associated with several pathophysiological actions that facilitate specific tissue and organ injuries including the production of cell proliferation, pro-inflammatory mediators, extracellular matrix synthesis and release of reactive oxygen species. 9 In this regard, the strategy to inhibit the production of Ang II by inhibiting ACE or RAS metabolism seems to be acceptable in improving elevated BP. In fact, captopril (a potent peptidic ACE inhibitor) was first developed as a therapeutic drug. The successful development of captopril for hypertension treatment 10 also provides us with the possibility of BP modulation by small peptides, because captopril’s structure to bind to the active site of ACE with high affinity is based on the mother peptide Ala-Pro (Phe-Ala-Pro for enalapril). 2 In the field of food sciences, numerous ACE inhibitory peptides have been isolated from the digestion or enzymatic hydrolysis of natural resources.
Natural resources that can provide ACE inhibitory peptides are sardine, 11 fermented milk, 12 casein, 13 royal jelly, 14 bonito, 15 sesame, 16 Mycoleptodonoides aitchisonii, 17 Wakame 18 and Nori (Porphya yezoensis) 19 were found to be good FOSHU sources in Japan. According to many ACE inhibition studies, most natural resources rich in proteins or traditional fermented foods such as tuna, 20 wheat, 21 procine, 22 mushroom, 23 cotton leafworm, 24 salt-free soy sauce, 25 Chungkookjang 26 or fermented soybean paste 27 seem to be good ACE inhibitory contributors. Taking into consideration that ACE has four functional amino acid residues of Tyr, Arg, Glu and Lys at the active site, and three hydrophobic binding subsites, 28 favorable blockade of ACE action would be primarily achieved by smaller di- or tripeptides. ACE inhibitory peptides (> 400) reported so far 2 provide the prevalence that small peptides with hydrophobic and aromatic amino acid residues such as Tyr, Phe, Trp and Pro at the C-terminal 29 have a potent ability to inhibit ACE activity with an IC 50 value of <100 µM. However, such ACE inhibitory peptides show poor inhibitory activity at the micro-M order compared to the nano-M order of therapeutic drugs. Therefore, it has become unclear whether the intake of ACE inhibitory peptides can exert a BP lowering effect in vivo via the suppression of circulating RAS.

Antihypertensive Effect of ACE Inhibitory Peptides


Information of dosage is also listed. It is clear that small peptides can exert an antihypertensive action following oral administration to mild hypertensive subjects, similar to therapeutic ACE inhibitory drugs, despite their poor potency on ACE inhibitory activity. It seems likely that ca. 20-fold higher doses of small peptides than drugs are required to exert significant BP lowering effects in spontaneously hypertensive rats (SHR). 30 The higher dosage of antihypertensive peptides could be partly understood by their lower ACE inhibitory activities.
Our recent studies, however, have provided alternative evidence that oral administration of antihypertensive VY to SHR 31 or to Tsukuba hypertensive mice 32 bearing human RAS did not alter plasma ACE activity at all. Similar findings with no influence of plasma ACE activity have also been reported in the oral intakes of egg white protein hydrolysate 33 and sour milk peptides. 34 These unexpected results led us to further investigation regarding antihypertensive mechanism(s) of small bioactive peptides and will be seen in the next section.

  • Regulation of Vessel Function by Peptides


Vascular Relaxation by Peptides


It has been clarified that absorbed VY was accumulated in the kidney and aorta in orally administrated SHR, along with the suppression of local ACE activities in both organs. 31 This finding suggested that both organs must be a targeted tissue for antihypertensive VY. The report that therapeutic ACE inhibitor captopril controlled vessel functions 35 also allowed us to consider the potential role of ACE inhibitory small peptides in contracted vessels.
Some peptides such as VY, Trp-His (WH), 6 Arg-Ala-Asp-His-Pro (-Phe) (RADHP(F)), 36,37 carnosine (β-Ala-His), 38 and Tyr-Pro-Ile (YPI) 36 have already been identified as a vasoactive (vasorelaxant) peptide in contracted aorta. Thus far, it is clear that various small peptides evoke a vasorelaxation effect. 6, the strength of a peptide to relax contracted aorta largely differs from their peptide sequence. These reported vasoactive peptides can be classified into two categories, i.e. endothelium-dependent and endothelium-independent types. Endothelium-dependent vasorelaxation is achieved by mainly cyclooxygenase (COX)/ cyclic adenosine monophosphate (cAMP), nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) and endothelium-derived hyperpolarizing factor (EDHF) systems including the bradykinin receptor signaling pathway. 39 Endothelial COX pathway mediates the production of prostaglandin I 2 (PGI 2 ) and relaxes vascular smooth muscle cells (VSMC) via the stimulation of prostacyclin receptor/cAMP-cAMP-dependent protein kinase A (PKA) pathway.
Bradykinin or EDHF system is also involved in the vasorelaxation signal talk by epoxyeicosatrienoic acids (EETs)-induced activation of BK Ca (large conductance Ca 2+ -activated K + channels). NO is a well-known vasorelaxant gas by endothelial NO synthase (eNOS) and in part by bradykinin stimulation. 40 The NO relaxation is caused by cytosolic soluble guanylyl cyclase (sGC) and the subsequent cGMP-dependent protein kinase (PKG) activation. The vasorelaxation of RADHP is due to a stimulation of bradykinin B1-receptor 36 to enhance NO/cGMP pathway, and the vasorelaxation of Met-Tyr (MY) from sardine muscle hydrolysate is caused by carbon monoxide (CO) production via heme oxygenase-1 (HO-1) activation, which stimulates cGMP production. 41 However, less useful studies regarding factors responsible for vasoactive peptides for endothelium-dependent action have yet to be conducted. So far, there were no reports on endothelium-independent peptides, except for carnosine (β-Ala-His), by which cGMP production was stimulated in VSMC. 
38, endothelium-independent or smooth muscle-dependent vasorelaxation can be achieved by the suppression of receptor-related signaling pathways through AT 1 -R, α-adrenergic and endothelin-1 receptors. 42,43 The activation of cGMP pathway or subsequent hyperpolarization by K + channels also induces vasorelaxation by lowering intracellular Ca 2+ concentration ([Ca 2+ ] i ) through a voltage-dependent (i.e. voltage-gated)  L -type Ca 2+ channel (VDCC). The mechanisms underlying vasorelaxation of small peptides will be seen in the next section, but we can propose structural factors of peptides eliciting endothelium-independent vasorelaxation. It is probable that WH (or HW) and HRW are the most effective sequences showing endothelium-independent vasorelaxation. The importance of the peptide backbone can be also understood for vasorelaxation as corresponding amino acids were no longer vasoactive. 4 The zero effect of each amino acid in vasorelaxant HRW suggested that Arg in HRW did not act as a NO donor. 
44 The positively charged guanidinium group (pKa: 12.5) of Arg residue at the assayed conditions may play a role in exerting the endothelium-independent vasorelaxation of HRW, since His-citrulline-Trp (H-Cit-W) significantly lost the power of mother peptide HRW. Methylated HRWs at either π or τ position of imidazole group, which led to a decrease of the activity of mother HRW, also provided useful information that the imidazole proton is a key factor for vasorelaxation. A significant loss of vasorelaxation power of Trp-Arg-His (WRH), a reverse sequence of HRW, indicates that along with amino acid compositions, the sequence of tripeptide manages to enhance vasorelaxation power. The structural factors of small peptides responsible for endothelium-independent relaxation can be summarized as follows: 
(1)  L -configuration of peptide sequence, 
(2) basic amino acids (preferably protonated residue) and 
(3) nitrogen-containing or aromatic amino acids at C-/N-terminals. 
Longer peptide length is not necessarily essential for endothelium-independent relaxation.

Vasorelaxation Mechanisms of Peptides


Useful information on the underlying vasorelaxation mechanism of endothelium-independent small peptides has been reported; a vasorelaxant dipeptide VY showed a significant antiproliferative action against VSMC. The effect was observed in both Ang II- and Bay K8644-stimulated VSMC, independent of ACE inhibition and Ang II-related receptors. In particular, inhibition of Bay K8644-stimulated VSMC proliferation indicates the involvement of VY in VDCC, since Bay K8644 is an agonist of VDCC. Based on the interesting finding, the action of potent vasoactive dipeptide WH was deeply investigated as a measure of [Ca 2+ ] i through VDCC. 45, a direct monitoring of [Ca 2+ ] i in Fura-2/AM (a Ca 2+ -specific fluorescent probe)-loaded VSMC indicates that WH has an ability to inhibit [Ca 2+ ] i elevated by Ang II via the stimulation of diverse pathways as mentioned above. 42,43 Further examination of CaCl 2 -stimulated [Ca 2+ ] i increase on VSMC in Ca 2+ -free buffer, in which [Ca 2+ ] i increase is restrictive to extracellular Ca 2+ route into VSMC, demonstrated that the lowering of elevated [Ca 2+ ] i by WH was in part caused by the inhibition of extracellular Ca 2+ influx across VSMC membrane. 
The observation that Bay K8644-induced elevation of [Ca 2+ ] i was inhibited by WH also allowed us to understand the possible involvement of WH in the binding of VDCC proteins. Although no useful information was obtained for VDCC blocking action of small peptides, some challenging studies have been attempted to clarify the relationship between VDCC proteins and blockers. Successful results were obtained by photoaffinity labeling methods 46,47 for distinct VDCC binding sites of phenylalkylamine (PAA)-type (e.g. verapamil) and dihydropyridine (DHP)-type (e.g. nifedipine and nimodipine). Namely, according to the photoaffinity labeling studies, it is clear that DHP-type blockers bind favorably to the extracellular side of VDCC proteins located at the loop connecting segments between S5–S6 on domain III and the end segment of S6 on domain IV in the α 1 -subunit. 46 In contrast, PAA-type blockers bind to an intracellular side of VDCC proteins located at the end of helix of S6 on domain IV in α 1 -subunit. 47 On the basis of these findings, combination experiments of WH with DHP- or PAA-type blockers were performed to clarify the potential role of small peptides including WH in VDCC proteins. 
The WH-induced [Ca 2+ ] i reduction in Bay K8644-stimulated VSMC was significantly abolished by verapamil, while small enhancement of the power was observed in the presence of nifedipine. 45 The study also revealed that the [Ca 2+ ] i reduction potential of nifedipine was abolished by verapamil, while nimodipine as a DHP-type blocker did not alter. It has been reported that the binding of PAA-type blockers to intracellular site of VDCC was apparently inhibited by DHP-type blockers via an allosteric mechanism, such that the DHP-type blocker could not bind to the extracellular site of VDCC. 48–52 Taken together, WH or other endothelium-independent vasorelaxant small peptides would play a role in the regulation of [Ca 2+ ] i in VSMC by their binding to the extracellular site of VDCC similar to DHP-type blockers. 
Structural factors for VDCC blocking action by peptides are also under consideration from the viewpoint of VDCC blockers. Amlodipine (pKa = 8.6), a VDCC blocker, significantly lost the activity when experimental pH was changed from 6.0 to 10.0. 53 Nifedipine, a DHPtype blocker, the N1 hydrogen atom of the heterocyclic pyridine ring has a key role in the binding to VDCC α 1 s subunit as a proton donor of hydrogen bond to Gln 1060 residue. 54 This indicates that the imidazole proton of His residue (pKa = 6.04) and/or neutral indole group of Trp residue may play an important role in the binding of peptides to the extracellular site of VDCC. Based on this structural role, more potent peptides must be clarified for further study.

Anti-atherosclerotic Effect of Peptide


Vasorelaxant small peptides such as WH and HRW via suppression of [Ca 2+ ] i through VDCC blocking action may be of great benefit to prevent or improve hypertension and related cardiovascular diseases, since the onset of vessel diseases such as atherosclerosis is promoted by the proliferation and/or migration of VSMC. 43 A challenging study on the anti-atherosclerosis effect of VDCC blocker-like small peptides (WH) firstly demonstrated that a 9-week administration of WH (10 or 100 mg/kg/ day) to apolipoprotein (apo) E-deficient mice could suppress the atherosclerotic lesion without any alteration on lipid profiles. 
Between both WH groups, no significant dose-dependent reduction of the area was observed, suggesting that a WH diet at a dose of 10 mg/kg/day may be enough to elicit the effect. Asimilar effect was observed for VDCC blockers, amlodipine 55 and azelnidipine, 56 in apo E-deficient mice, which strongly suggested that the blockade of extracellular Ca 2+ influx by any VDCC blocking peptides would be sufficient to improve vessel diseases.

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