Positive Charges Of Lysine Residues Research Articles

Dopamine-Substituted Multidomain Peptide Hydrogel with Inherent Antimicrobial Activity and Antioxidant Capability for Infected Wound Healing.

Wound infection can cause a delay in wound healing or even wound deterioration, threatening patients' lives. The excessive accumulation of reactive oxygen species (ROS) in infected wounds activates a strong inflammatory response to delay wound healing. Therefore, it is highly desired to develop hydrogels with inherent antimicrobial activity and antioxidant capability for infected wound healing. Herein, a dopamine-substituted multidomain peptide (DAP) with inherent antimicrobial activity, strong skin adhesion, and ROS scavenging has been developed. DAP can form bilayer β-sheets with dopamine residues on the surface of nanofibers. The enhanced rheological properties of DAP-based hydrogel can be achieved not only through UV irradiation but also by incorporation of multivalent ions (e.g., PO43-). Furthermore, the DAP hydrogel shows a broad spectrum of antimicrobial activity due to the high positive charges of lysine residues and the β-sheet formation. When applied to full-thickness dermal wounds in mice, the DAP hydrogel results in a significantly shortened inflammatory stage of the healing process because of its remarkable antimicrobial activity and antioxidant capability. Accelerated wound closure with thick granulation tissue, uniform collagen arrangement, and dense vascularization can be achieved. This work suggests that the DAP hydrogel can serve as antimicrobial coating and ROS-scavenging wound dressing for bacterial-infected wound treatment.

Label-Free Monitoring of Histone Acetylation Using Aptamer-Functionalized Field-Effect Transistor and Quartz Crystal Microbalance Sensors.

Chemical and enzymatic modifications of amino acid residues in protein after translation contain rich information about physiological conditions and diseases. Histone acetylation/deacetylation is the essential post-translational modification by regulating gene transcription. Such qualitative changes of biomacromolecules need to be detected in point-of-care systems for an early and accurate diagnosis. However, there is no technique to aid this issue. Previously, we have applied an aptamer-functionalized field-effect transistor (FET) to the specific protein biosensing. Quantitative changes of target protein in a physiological solution have been determined by detecting innate charges of captured protein at the gate-solution interface. Moreover, we have succeeded in developing an integrated system of FET and quartz crystal microbalance (QCM) sensors for determining the adsorbed mass and charge, simultaneously or in parallel. Prompted by this, in this study, we developed a new label-free method for detecting histone acetylation using FET and QCM sensors. The loss of positive charge of lysine residue by chemically induced acetylation of histone subunits (H3 and H4) was successfully detected by potentiometric signals using anti-histone aptamer-functionalized FET. The adsorbed mass was determined by the same anti-histone aptamer-functionalized QCM. From these results, the degree of acetylation was correlated to the charge-to-mass ratio of histone subunits. The histone required for the detection was below 100 nM, owing to the high sensitivity of aptamer-functionalized FET and QCM sensors. These findings will guide us to a new way of measuring post-translational modification of protein in a decentralized manner for an early and accurate diagnosis.

Human Apurinic/Apyrimidinic Endonuclease (APE1) Is Acetylated at DNA Damage Sites in Chromatin, and Acetylation Modulates Its DNA Repair Activity.

ABSTRACTApurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme that repairs AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of the positive charges of the lysine residues in the N-terminal domain of APE1 induces a conformational change; this in turn enhances the AP endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus, mammalian cells, unlike Saccharomyces cerevisiae or Escherichia coli cells, require acetylation of APE1 for the efficient repair of AP sites and base damage in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity.

Role of positive charge of lysine residue on cytochrome c3for electrostatic interaction with hydrogenase

Cytochrome c3from Desulfovibrio vulgaris (Miyazaki) is an electron transfer protein containing four hemes per molecule. Its physiological electron transfer partner is the hydrogenase which catalyzes reversible oxidation of hydrogen. The complex formation between cytochrome c3and hydrogenase is caused by electrostatic interaction, because cytochrome c3is a basic protein and hydrogenase is an acidic protein. As cytochrome c3has 20 lysine residues among 108 amino acids, the positive charges of some lysine residues may play an important role in the interaction with hydrogenase. To clarify the role of positive charge of lysine residue, the positive charge was changed to neutral or negative charge using chemical modification and site-directed mutagenesis. When the positive charges around heme IV were changed, the hydrogen evolution rate with hydrogenase decreased. The affinity between hydrogenase and mutated cytochrome c3(K57Q, K57E, K72Q, K94Q, K94E) were not affected. On the other hand, the affinity of K72E cytochrome c3for hydrogenase was very low. These results suggest that the positive charge around heme IV plays an important role in the electrostatic interaction with hydrogenase in hydrogen evolution.

Roles of peptide–peptide charge interaction and lipid phase separation in helix–helix association in lipid bilayer

The roles of peptide–peptide charged interaction and lipid phase separation in helix–helix association in lipid bilayers were investigated using a model peptide, P 24, as a transmembrane α-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P 24W) and pyrenylalanine (P 24Pya), were introduced into the sequence of P 24, respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P 24W and pyrenylalanine in P 24Pya or excimer formation between P 24Pya themselves. To evaluate the effect of charged interaction on the association between α-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P 24EW) and lysine (P 24KPya), were introduced into P 24W and P 24Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P 24KPya and the negative charge of glutamic acid residue in P 24EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P 24Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the α-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.

Hormonotoxins: the role of positive charge of lysine residue on the immunological, biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates.

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occurred through the epsilon-NH2 groups of alpha-subunit of oLH as judged from RP-HPLC analysis. A1:1 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.

Interaction of type I collagen fibrils with phospholipid vesicles

Type I collagen fibrils interact with phosphatidylcholine and phosphatidylglycerol vesicles. Fluorescence polarization of (1,6-diphenyl-1,3,5-hexatriene) DPH-labeled vesicles, circular dichroism and differential scanning calorimetry studies have been performed. The protein-lipid interaction produces a decrease of the enthalpy of the phospholipid phase transition. Positive charges of lysine residues of the protein are involved in the interaction as experiments with succinylated collagen show. The kinetic parameters and the extent of the fibrillogenesis of collagen are modified by the phospholipid vesicles.