[Arctigenin mitigates vascular endothelial injury in rats with pregnancy-induced hypertension via autophagy-NLRP3 inflammasome pathway].

This study aims to investigate the effect and mechanism of arctigenin(ARC) in the treatment of vascular endothelial injury in rats with pregnancy-induced hypertension(PIH). Fifty SD rats pregnant for 12 days were randomly assigned into a control group, a model group, an ARC group, a rapamycin(RAP, autophagy inducer) group, and an ARC+3-methyladenine(3-MA, autophagy inhibitor) group, with 10 rats in each group. The rats in the other groups except the control group were intraperitoneally injected with nitrosyl-L-arginine methyl ester(50 mg·kg~(-1)·d~(-1)) to establish the PIH model on the 13th day of pregnancy. On the 15th day of pregnancy, the rats in ARC, RAP, and ARC+3-MA groups were intraperitoneally injected with ARC(50 mg·kg~(-1)·d~(-1)), RAP(1 mg·kg~(-1)·d~(-1)), and 3-MA(15 mg·kg~(-1)·d~(-1))+ARC(50 mg·kg~(-1)·d~(-1)), respectively. The pregnant rats in the control group and the model group were intraperitoneally injected with the same amount of normal saline. The blood pressure and 24 h urine protein(24 h-UP) of pregnant rats in each group were measured before and after intervention. Cesarean section was performed to terminate pregnancy on day 21, and the body weight and body length of fetal rats were compared among groups. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes of placenta. The expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in placenta was detected by immunohistochemistry. The serum levels of ET-1 and nitric oxide(NO) were determined with corresponding kits. The expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein with CARD domain(ASC), caspase-1, interleukin(IL)-1ß, and IL-18 was determined by immunofluorescence and Western blot. The level of reactive oxygen species(ROS) in placenta was measured by fluorescence staining. The results showed that on day 12 of pregnancy, the blood pressure and 24 h-UP had no significant differences among groups. On days 15, 19, and 21, the blood pressure and 24 h-UP in the model group were higher than those in the control group(P<0.05). On days 19 and 21, the blood pressure and 24 h-UP in ARC group and RAP group were lower than those in the model group(P<0.05), and they were higher in the ARC+3-MA group than in the ARC group(P<0.05). On day 21, the model group had lower body weight and body length of fetal rats(P<0.05), higher serum level of ET-1, and lower serum level of NO(P<0.05) than the control group. Moreover, the placental tissue showed typical pathological damage, down-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and eNOS(P<0.05), up-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1ß, and IL-18(P<0.05), and elevated ROS level. Compared with the model group, ARC and RAP groups showed increased body weight and body length of fetal rats(P<0.05), lowered serum level of ET-1, elevated serum level of NO(P<0.05), reduced pathological damage of placental tissue, up-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and eNOS(P<0.05), down-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1ß, and IL-18(P<0.05), and lowered ROS level. Compared with ARC group, 3-MA reversed the effects of ARC on the above indicators. In conclusion, ARC can inhibit the activation of NLRP3 inflammasome and mitigate vascular endothelial damage in PIH rats by inducing autophagy of vascular endothelial cells.

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.

Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity.

It has been reported that the (6)CPCP(9) motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the (6)CPCP(9) motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the (6)CPCP(9) tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the (6)CPCP(9) motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the (6)CPCP(9) motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein's bioactivity.

Forced unfolding of apocytochrome b5 by steered molecular dynamics simulation.

Apocytochrome b5 (apocyt b5), a small b-type cytochrome with heme prosthetic group removal, has been subjected to steered molecular dynamics (SMD) simulations for investigating the consequences of mechanical force-induced unfolding. Both constant velocity (0.5 and 1.0 A/ps) and constant force (500, 750 and 1000 pN) stretching have been employed to model forced unfolding of apocyt b5. The results of SMD simulations elucidate that apocyt b5 is protected against external stress mainly through the interstrand hydrogen bonding between its beta1-beta2 and beta2-beta3 strands, highlighting the importance of hydrophobic core 2 in stabilization of apocyt b5. The existence of intermediate states manifested by current simulations in the forced unfolding pathway of apocyt b5 is different from the observations in pervious thermal or chemical unfolding studies in the absence of force. The present study could thus provide insights into the relationship between the two cooperative functional modules of apocyt b5 and also guide the rational molecular design of heme proteins.

Structural prediction of the beta-domain of metallothionein-3 by molecular dynamics simulation.

The beta-domain of metallothionein-3 (MT3) has been reported to be crucial to the neuron growth inhibitory bioactivity. Little detailed three-dimensional structural information is available to present a reliable basis for elucidation on structure-property-function relationships of this unique protein by experimental techniques. So, molecular dynamics simulation is adopted to study the structure of beta-domain of MT3. In this article, a 3D structural model of beta-domain of MT3 was generated. The molecular simulations provide detailed protein structural information of MT3. As compared with MT2, we found a characteristic conformation formed in the fragment (residue 1-13) at the N-terminus of MT3 owing to the constraint induced by 5TCPCP9, in which Pro7 and Pro9 residues are on the same side of the protein, both facing outward and the two 5-member rings of prolines are arranged almost in parallel, while Thr5 is on the opposite side. Thr5 in MT3 is also found to make the first four residues relatively far from the fragment (residue 23-26) as compared with MT2. The simulated structure of beta-domain of MT3 is looser than that of MT2. The higher energy of MT3 than that of MT2 calculated supports these conclusions. Simulation on the four isomer arising from the cis- or trans-configuration of 6CPCP9 show that the trans-/trans-isomer is energetic favorable. The partially unfolding structure of beta-domain of MT3 is also simulated and the results show the influence of 6CPCP9 sequence on the correct folding of this domain. The correlations between the bioactivity of MT3 and the simulated structure as well as the folding of beta-domain of MT3 are discussed based on our simulation and previous results.

Neuronal growth-inhibitory factor (metallothionein-3): structure-function relationships.

Neuronal growth-inhibitory factor (GIF), also named metallothionein-3, inhibits the outgrowth of neuronal cells. Recent studies on the structure of human GIF, carried out using NMR and molecular dynamics simulation techniques, have been summarized. By studying a series of protein-engineered mutants of GIF, we showed that the bioactivity of GIF is modulated by multiple factors, including the unique TCPCP motif-induced characteristic conformation, the solvent accessibility and dynamics of the metal-thiolate cluster, and the domain-domain interactions.

The role of Ile476 in the structural stability and substrate binding of human cytochrome P450 2C8.

The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown. Therefore, six site-directed mutants of CYP2C8 were constructed to better define this. By UV-visible and circular dichroism spectroscopy studies, we studied for the first time the structural stability and all-trans-retinoic acid binding capability of the CYP2C8 variants. We found that the ferric CYP2C8 went through three states during thermal unfolding. Combined with substrate binding studies, our data revealed that residue 476 was involved in contact with substrate and was important for maintaining the thermal stability of CYP2C8.

The structural and biological significance of the EAAEAE insert in the alpha-domain of human neuronal growth inhibitory factor.

Human neuronal growth inhibitory factor (hGIF) is able to inhibit the outgrowth of neurons. As compared with the amino acid sequences of metallothionein 1/2, hGIF contains two insertions: a Thr at position 5 and an acidic hexapeptide EAAEAE(55-60) close to the C-terminus. Moreover, all mammalian growth inhibitory factor sequences contain a conserved CPCP(6-9) motif. Previous studies have demonstrated that the TCPCP(5-9) motif is pivotal to its bioactivity, but no specific role has been assigned to the unique EAAEAE(55-60) insert. To investigate the potential structural and biological significance of the EAAEAE(55-60) insert, several mutants were constructed and investigated in detail. Notably, deletion of the acidic insert (the Delta55-60 mutant) reduced the inhibitory activity, whereas the bioactivities of other mutants did not change much. Then, spectroscopic characterization and molecular dynamics simulation were performed to investigate the potential causes of the reduced bioactivity of the Delta55-60 mutant. It was found that the domain-domain interaction mechanism of hGIF was different from that of metallothionein 2. It was also shown that the acidic insert could regulate the interdomain interactions in hGIF, leading to the structural change in the beta-domain, which resulted in the alteration of the solvent accessibility and metal release ability, thus playing an important role in the biological activity of hGIF. Our studies provided useful information on the domain-domain interaction at the molecular level for the first time, and shed new light on the mechanism of the bioactivity of hGIF.

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF).

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the (6)CPCP(9) motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.