In vitro and in silico studies of novel synthetic ACE-inhibitory peptides derived from Saccharomyces cerevisiae protein hydrolysate

The structure-function relation of YR-10 (YGKPVAVPAR) was investigated by synthesizing four structural analogs of that including YHR-10 (YGKHVAVHAR), GA-8 (GKPVAVPA), GHA-8 (GKHVAVHA), and PAR-3 (PAR). GA-8 (GKPVAVPA) was synthesized on the basis of simulated enzymatic gastrointestinal digestion performed by bioinformatics tools (expasy-peptide cutter). This study explains the molecular mechanisms for the interaction of synthetic peptides with ACE. The IC₅₀ values of each were 139.554 ± 2.3, 61.91 ± 1.2, 463.230 ± 3.56, 135.135 ± 2.1, 514.024 ± 5.86 µM, respectively. Results indicated that Pro replacement with His in YR-10 and GA-8 increased ACE inhibitory activity respectively, by 55.63% and 70.82%. Removal of Tyr and Arg from respectively N and C terminal positions of YR-10, following in silico simulated gastrointestinal digestion caused the 3.31 fold decrease in ACE inhibitory activity. YHR-10 showed the best docking poses, and GHA-8 exhibited interaction with Zn²⁺. Lineweaver–Burk plots of most active peptides suggest that they act as noncompetitive inhibitors against ACE.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

Thermodynamics of zinc binding to rhodanese

The binding of zinc ion (Zn²⁺) to rhodanese at two pH values was studied by microcalorimetry and the free energy, enthalpy, and entropy changes determined. Binding exhibited rather large endothermic enthalpy changes quite similar to those observed for zinc-model compound interactions. The large positive entropy changes which accompany binding appear to be a feature common to Zn²⁺-apocarbonic anhydrase systems as well. The correlations between Zn²⁺ interaction with model compounds resembling protein side chains and the thermodynamic values obtained for Zn²⁺-protein interactions suggest that endothermic enthalpies of binding should commonly be observed under slightly acidic to basic conditions. It is found that commercial rhodanese binds Zn²⁺ with moderate to weak affinity by a process that is entropy driven much like that of other Zn²⁺-protein interactions.     

Molecular Features of the Zn2+ Binding Site in the Prion Protein Probed by 113Cd NMR

The cellular prion protein (PrP^C) is a zinc-binding protein that contributes to the regulation of Zn²⁺ and other divalent species of the central nervous system. Zn²⁺ coordinates to the flexible, N-terminal repeat region of PrP^C and drives a tertiary contact between this repeat region and a well-defined cleft of the C-terminal domain. The tertiary structure promoted by Zn²⁺ is thought to regulate inherent PrP^C toxicity. Despite the emerging consensus regarding the interaction between Zn²⁺ and PrP^C, there is little direct spectroscopic confirmation of the metal ion’s coordination details. Here, we address this conceptual gap by using Cd²⁺ as a surrogate for Zn²⁺. NMR finds that Cd²⁺ binds exclusively to the His imidazole side chains of the repeat segment, with a dissociation constant of ～1.2 mM, and promotes an N-terminal-C-terminal cis interaction very similar to that observed with Zn²⁺. Analysis of ¹¹³Cd NMR spectra of PrP^C, along with relevant control proteins and peptides, suggests that coordination of Cd²⁺ in the full-length protein is consistent with a three- or four-His geometry. Examination of the mutation E199K in mouse PrP^C (E200K in humans), responsible for inherited Creutzfeldt-Jakob disease, finds that the mutation lowers metal ion affinity and weakens the cis interaction. These findings not only provide deeper insight into PrP^C metal ion coordination but they also suggest new perspectives on the role of familial mutations in prion disease.     

Ca-induced stimulation of the membrane binding of Escherichia coli SecA and its association with signal peptides of secretory proteins

Previously, it was found that Ca²⁺ stimulates the intrinsic Escherichia coli SecA ATPase activity [Kim et al., FEBS Lett. 493 (2001) 12-16]. Now, we suggest that Ca²⁺ is required for efficient interaction of SecA with membranes and the signal peptide of ribose-binding protein. When the amount of external Ca²⁺ was enhanced, the amounts of membrane-bound SecA and its lipid/ATPase activity increased. In the presence of entrapped Ca²⁺ in liposomes, the binding was also stimulated in a Ca²⁺ concentration-dependent manner. The effect of Ca²⁺ on the functional regulation of SecA was also evident in the presence of the signal peptides of secretory proteins, which the interaction of SecA with the signal peptide increased with increasing Ca²⁺ concentration in the presence of membranes. However, other divalent cations including Mg²⁺, Mn²⁺, and Zn²⁺ had inhibitory or no effect, suggesting a specific role of Ca²⁺ in SecA interaction with lipid bilayers and signal peptides.     

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn²⁺ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn²⁺-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn²⁺-binding motifs. Here, we show that Zn²⁺ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn²⁺ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn²⁺ on membrane repair is abolished in mg53^−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn²⁺ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn²⁺-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn²⁺ interaction with MG53 in protection against injury to the cell membrane.     

Purification and Characterization of an Alkaline Protease from the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺, Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1–10-phenanthroline, and iodoacetic acid. The K _m and V _max values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 μmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.     

Role of zinc and copper ions in the pathogenetic mechanisms of Alzheimer’s and Parkinson’s diseases

Disbalance of zinc (Zn²⁺) and copper (Cu²⁺) ions in the central nervous system is involved in the pathogenesis of numerous neurodegenerative disorders such as multisystem atrophy, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Wilson-Konovalov disease, Alzheimer’s disease, and Parkinson’s disease. Among these, Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most frequent age-related neurodegenerative pathologies with disorders in Zn²⁺ and Cu²⁺ homeostasis playing a pivotal role in the mechanisms of pathogenesis. In this review we generalized and systematized current literature data concerning this problem. The interactions of Zn²⁺ and Cu²⁺ with amyloid precursor protein (APP), β-amyloid (Abeta), tau-protein, metallothioneins, and GSK3β are considered, as well as the role of these interactions in the generation of free radicals in AD and PD. Analysis of the literature suggests that the main factors of AD and PD pathogenesis (oxidative stress, structural disorders and aggregation of proteins, mitochondrial dysfunction, energy deficiency) that initiate a cascade of events resulting finally in the dysfunction of neuronal networks are mediated by the disbalance of Zn²⁺ and Cu²⁺.     

A Brief Overview from the Physiological and Detrimental Roles of Zinc Homeostasis via Zinc Transporters in the Heart

Zinc (mostly as free/labile Zn²⁺) is an essential structural constituent of many proteins, including enzymes in cellular signaling pathways via functioning as an important signaling molecule in mammalian cells. In cardiomyocytes at resting condition, intracellular labile Zn²⁺ concentration ([Zn²⁺]_i) is in the nanomolar range, whereas it can increase dramatically under pathological conditions, including hyperglycemia, but the mechanisms that affect its subcellular redistribution is not clear. Therefore, overall, very little is known about the precise mechanisms controlling the intracellular distribution of labile Zn²⁺, particularly via Zn²⁺ transporters during cardiac function under both physiological and pathophysiological conditions. Literature data demonstrated that [Zn²⁺]_i homeostasis in mammalian cells is primarily coordinated by Zn²⁺ transporters classified as ZnTs (SLC30A) and ZIPs (SLC39A). To identify the molecular mechanisms of diverse functions of labile Zn²⁺ in the heart, the recent studies focused on the discovery of subcellular localization of these Zn²⁺ transporters in parallel to the discovery of novel physiological functions of [Zn²⁺]_i in cardiomyocytes. The present review summarizes the current understanding of the role of [Zn²⁺]_i changes in cardiomyocytes under pathological conditions, and under high [Zn²⁺]_i and how Zn²⁺ transporters are important for its subcellular redistribution. The emerging importance and the promise of some Zn²⁺ transporters for targeted cardiac therapy against pathological stimuli are also provided. Taken together, the review clearly outlines cellular control of cytosolic Zn²⁺ signaling by Zn²⁺ transporters, the role of Zn²⁺ transporters in heart function under hyperglycemia, the role of Zn²⁺ under increased oxidative stress and ER stress, and their roles in cancer are discussed.

     

Casein-Derived Peptides with Antihypertensive Potential: Production,Identification and Assessment of Complex Formation with Angiotensin I-Converting Enzyme (ACE) through Molecular Docking Studies

Hypertension is nowadays one of the major world concerns in public health. Several food proteins, among which caseins, can be substrates for generating peptides with antihypertensive potential. With the increasingly evolution of computational tools, in silico molecular modeling have gained prominence in studies of protein-ligand complexes in different research fields, such as pharmaceutics and biochemical engineering. However, the application of such methodologies in food-related research can be considered still embryonic. Thus, the central aim of the present work was to apply molecular modelling in order to elucidate the molecular bases of the anti-hypertensive potential of milk caseins-derived peptides. Firstly, hydrolysates obtained from a controlled trypsinolysis of caseins were fractioned according to their molecular weight, by ultrafiltration and RP-HPLC. The obtained fractions were evaluated with regard to their in vitro inhibitory angiotensin-converting enzyme activity (%I_ACE). Six chromatographic fractions were identified, and three of them displayed high ACE-inhibition (F₁: 80.68%; F₂: 79.00%; and F₄: 62.44%). Finally, intermolecular interactions networks in complexes formed between ACE and the identified peptides were assessed through in silico molecular docking. At the molecular level, a correlation between in vitro and in silico results was found: the peptides FFVAPFPEVFGK (F₆), FALPQYLK (F₂, F₄) and ALNEINQFYQK (F₁) presented the lowest biding energies and interacted by specific H-bonds, electrostatic and hydrophobic interactions formed within ACE active site S1 residues (Ala354, Glu384, and Tyr 523) and the Zn²⁺ coordinated residues (His383, His387, and Glu411). The fraction F₃, despite its low total peptide concentration, presented a moderate inhibitory activity for ACE (49.2%), likely due to H-bonds between HQGLPQEVLNENLLR and the active site S1 residues.

     

Membrane Interactions of S100A12 (Calgranulin C)

S100A12 (Calgranulin C) is a small acidic calcium-binding peripheral membrane protein with two EF-hand structural motifs. It is expressed in macrophages and lymphocytes and highly up-regulated in several human inflammatory diseases. In pigs, S100A12 is abundant in the cytosol of granulocytes, where it is believed to be involved in signal modulation of inflammatory process. In this study, we investigated the interaction of the porcine S100A12 with phospholipid bilayers and the effect that ions (Ca²⁺, Zn²⁺ or both together) have in modifying protein-lipid interactions. More specifically, we intended to address issues such as: (1) is the protein-membrane interaction modulated by the presence of ions? (2) is the protein overall structure affected by the presence of the ions and membrane models simultaneously? (3) what are the specific conformational changes taking place when ions and membranes are both present? (4) does the protein have any kind of molecular preferences for a specific lipid component? To provide insight into membrane interactions and answer those questions, synchrotron radiation circular dichroism spectroscopy, fluorescence spectroscopy, and surface plasmon resonance were used. The use of these combined techniques demonstrated that this protein was capable of interacting both with lipids and with ions in solution, and enabled examination of changes that occur at different levels of structure organization. The presence of both Ca²⁺ and Zn²⁺ ions modify the binding, conformation and thermal stability of the protein in the presence of lipids. Hence, these studies examining molecular interactions of porcine S100A12 in solution complement the previously determined crystal structure information on this family of proteins, enhancing our understanding of its dynamics of interaction with membranes.     

Mechanism of Free Zn(2+) Enhancing Inhibitory Effects of EGCG on the Growth of PC-3 Cells: Interactions with Mitochondria

Green tea and its major constituent epigallocatechin gallate (EGCG) are known for their chemopreventive effects including those against prostate cancer, which could be mediated by metal ions. Zn²⁺ is an essential trace element that is required for human health and plays an important role in the normal function of the prostate gland. In the present study, the effect of EGCG on cell membrane and mitochondria of PC-3 (prostate carcinoma) cells in the presence and absence of Zn²⁺ was studied. These studies revealed that EGCG, Zn²⁺, or EGCG + Zn²⁺ affected the morphology of PC-3 cells and induced apoptosis in PC-3 cells. It was observed that effects of treatment with EGCG, Zn²⁺, or EGCG + Zn²⁺on mitochondria showed EGCG + Zn²⁺ > Zn²⁺ > EGCG, including cytochrome C release from the intermembrane space into the cytosol, inhibited the synthesis of ATP, loss of mitochondrial membrane potential, and activation of caspase-9. However, the order of effect on depressing membrane fluidity of PC-3 cells was EGCG > EGCG + Zn²⁺ > Zn²⁺. In summary, these findings suggest that EGCG, Zn²⁺, and EGCG + Zn²⁺ induce necrosis or apoptosis of PC-3 cells through mitochondria-mediated apoptotic pathway and free Zn²⁺-enhanced effects of EGCG on PC-3 cells due to its interactions with mitochondria.     

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

This report demonstrates that transition metal ions and selenite affect the arsenite methylation by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) in vitro. Co²⁺, Mn²⁺, and Zn²⁺ inhibited the arsenite methylation by hAS3MT in a concentration-dependent manner and the kinetics indicated Co²⁺ and Mn²⁺ to be mixed (competitive and non-competitive) inhibitors while Zn²⁺ to be a competitive inhibitor. However, only a high concentration of Fe²⁺ could restrain the methylation. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and hAS3MT. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the transition metal ion or selenite inhibition of hAS3MT activity. The inhibition of arsenite methylating activity of hAS3MT by selenite was reversed by 2 mM DTT (dithiothreitol) but neither by cysteine nor by β-mercaptoethanol. Whereas, besides DTT, cysteine can also prevent the inhibition of hAS3MT activity by Co²⁺, Mn²⁺, and Zn²⁺. Free Cys residues were involved in the interactions of transition metal ions or selenite with hAS3MT. It is proposed that the inhibitory effect of the ions (Co²⁺, Mn²⁺, and Zn²⁺) or selenite on hAS3MT activity might be via the interactions of them with free Cys residues in hAS3MT to form inactive protein adducts.     

SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2′ cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca²⁺, but that of SARS-2 shows a greater response to the presence of Ca²⁺. Both FPs bind two Ca²⁺ ions as does SARS-1 FP, but the two Ca²⁺ binding sites of SARS-2 exhibit greater cooperativity. This Ca²⁺ dependence by the SARS-2 FP is very ion-specific. These results show that Ca²⁺ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca²⁺ channel.     

Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn²⁺ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn²⁺-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn²⁺-induced agglutination. In contrast with Hg²⁺ and Cd²⁺ ions, Zn²⁺ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn²⁺ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn²⁺ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure     

Negatively charged residues in the membrane ordering activity of SARS-CoV-1 and -2 fusion peptides

Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus (“SARS-1”) and severe acute respiratory syndrome coronavirus-2 (“SARS-2”) using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca²⁺-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca²⁺. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP’s membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca²⁺ ions: its two segments FP1 and FP2 each bind one Ca²⁺. The binding of Ca²⁺, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca²⁺ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS-1 and SARS-2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.     

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (π_e) were clearly lower than the biological membrane pressure (24–30 mN m^− 1) and the HIV-1 FP (35 mN m^− 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G^E). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G^E values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP–lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.     

The Effects of Metal Ions on the Cytotoxicity and Selectivity of a Histidine-Containing Lytic Peptide

The histidine-containing peptide L5C (PAWRHAFHWAWHMLHKAA) is a histidine-rich lytic peptide. Interactions of some divalent metal ions with peptide L5C and their effects on the cell lysis activity of the peptide were studied. The presence of Cu²⁺ caused a secondary structure change (from random coil to α-helix) which resulted in the loss of cell lysis activity in peptide L5C. Binding of Zn²⁺ to peptide L5C also reduced the lytic activity of the peptide but Zn²⁺ did not affect the secondary structure of the peptides. Instead, Zn²⁺ induced peptide L5C aggregation. Unlike Zn²⁺ and Cu²⁺, Mg²⁺ had no significant effect on the activity of peptide L5C. Further experiments revealed that formed ion-peptide L5C complexes were sensitive to pH and dissociated in acidic solutions. Peptide L5C demonstrated improved pH-selectivity in the presence of trace amount of Zn²⁺. This property of histidine-containing lytic peptides can be used to improve their therapeutic effectiveness in the treatment of cancers.     

The effect of Zn2+ on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies

We studied the effects of Zn²⁺ on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn²⁺ inactivated the activity of PSCK (IC₅₀?=?.079?±?.004?mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn²⁺ induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn²⁺ directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn²⁺ bind to PSCK and one Zn²⁺ is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn²⁺ with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn²⁺ on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.