Thr-Arg

To prepare peptides with high angiotensin-converting enzyme (ACE) inhibitory (ACEi) activity, Alcalase was screened from five proteases and employed to prepare protein hydrolysate (TMH) of skipjack tuna (Katsuwonus pelamis) milts. Subsequently, 10 novel ACEi peptides were isolated from the high-ACEi activity TMH and identified as Tyr-Asp-Asp (YDD), Thr-Arg-Glu (TRE), Arg-Asp-Tyr (RDY), Thr-Glu-Arg-Met (TERM), Asp-Arg-Arg-Tyr-Gly (DRRYG), Ile-Cys-Tyr (ICY), Leu-Ser-Phe-Arg (LSFR), Gly-Val-Arg-Phe (GVRF), Lys-Leu-Tyr-Ala-Leu-Phe (KLYALF), and Ile-Tyr-Ser-Pro (IYSP) with molecular weights of 411.35, 404.41, 452.45, 535.60, 665.69, 397.48, 521.61, 477.55, 753.91, and 478.53 Da, respectively. Among them, the IC50 values of ICY, LSFR, and IYSP on ACE were 0.48, 0.59, and 0.76 mg/mL, respectively. The significant ACEi activity of ICY, LSFR, and IYSP with affinities of -7.0, -8.5, and -8.3 kcal/mol mainly attributed to effectively combining with the ACEi active sites through hydrogen bonding, electrostatic force, and hydrophobic interaction. Moreover, ICY, LSFR, and IYSP could positively influence the production of nitric oxide (NO) and endothelin-1 (ET-1) secretion in human umbilical vein endothelial cells (HUVECs) and weaken the adverse impact of norepinephrine (NE) on the production of NO and ET-1. In addition, ICY, LSFR, and IYSP could provide significant protection to HUVECs against H2O2 damage by increasing antioxidase levels to decrease the contents of reactive oxide species and malondialdehyde. Therefore, the ACEi peptides of ICY, LSFR, and IYSP are beneficial functional molecules for healthy foods against hypertension and cardiovascular diseases.

Selenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or Drosophila leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the inhibition of cell growth/proliferation, the accumulation of hydrogen peroxide in mammals and an unidentified reactive oxygen species (ROS) in Drosophila, and the activation of a defense system. Hydrogen peroxide accumulation in SEPHS1-deficient cells is mainly caused by the down-regulation of genes involved in ROS scavenging, and leads to the inhibition of cell proliferation and survival. However, the mechanisms underlying SEPHS1 regulation of redox homeostasis are still not understood.

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ~1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIaHis in isolation. The βFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.