NMR Structural Study of Syndecan-4 Transmembrane Domain with Cytoplasmic Region.

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.

Structural Studies of Expressed tIK, Anti-Inflammatory Peptide.

Cytokine imbalance is one of the causes of inflammation. Inflammation has yet to be adequately treated without side effects. Therefore, we tried to develop a peptide drug with minimal side effects. Peptide drugs have the advantage of being bio-friendly and bio-specific. In a previous study, three peptides with anti-inflammatory activity were derived based on a truncated IK (tIK) protein, which was a fragment of the IK protein with anti-inflammatory effects. The objective of this study was to optimize the process of expressing, isolating, and purifying the three peptides using bacterial strains and describe the process. Circular dichroism and solution state nuclear magnetic resonance spectroscopy were performed on the final purified high-purity peptide and its secondary structure was also identified.

NMR Studies of the Ion Channel-Forming Human Amyloid-ß with Zinc Ion Concentrations.

Alzheimer's disease (AD) is classified as an amyloid-related disease. Amyloid beta (Aß) is a transmembrane protein known to play a major role in the pathogenesis of AD. These Aß proteins can form ion channels or pores in the cell membrane. Studies have elucidated the structure of the transmembrane domain of Aß ion channels. In addition, various studies have investigated substances that block or inhibit the formation of Aß ion channels. Zinc ions are considered as potential inhibitors of AD. In this study, we focused on the transmembrane domain and some external domains of the Aß protein (hAPP-TM), and solution-state NMR was used to confirm the effect on residues of the protein in the presence of zinc ions. In addition, we sought to confirm the structure and orientation of the protein in the presence of the bicelle using solid-state NMR.

Structural and Mechanismic Studies of Lactophoricin Analog, Novel Antibacterial Peptide.

Naturally derived antibacterial peptides exhibit excellent pharmacological action without the risk of resistance, suggesting a potential role as biologicals. Lactophoricin-I (LPcin-I), found in the proteose peptone component-3 (PP3; lactophorin) of bovine milk, is known to exhibit antibiotic activity against Gram-positive and Gram-negative bacteria. Accordingly, we derived a new antibacterial peptide and investigated its structure-function relationship. This study was initiated by designing antibacterial peptide analogs with better antibacterial activity, less cytotoxicity, and shorter amino acid sequences based on LPcin-I. The structural properties of antibacterial peptide analogs were investigated via spectroscopic analysis, and the antibacterial activity was confirmed by measurement of the minimal inhibitory concentration (MIC). The structure and mechanism of the antibacterial peptide analog in the cell membrane were also studied via solution-state nuclear magnetic resonance (NMR) and solid-state NMR spectroscopy. Through 15N one-dimensional and two-dimensional NMR experiments and 31P NMR experiments, we suggest the 3D morphology and antibacterial mechanism in the phospholipid bilayer of the LPcin analog. This study is expected to establish a system for the development of novel antibacterial peptides and to establish a theoretical basis for research into antibiotic substitutes.

Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor.

BACKGROUND: Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects. OBJECTIVE: In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles. METHODS: The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2. RESULTS: The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal. CONCLUSION: We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

NMR structural studies and mechanism of action of Lactophoricin analogs as antimicrobial peptides.

Antimicrobial peptides (AMPs) are effective alternatives to conventional antibiotics. They protect the host from the constant invasion of a broad range of infectious microorganisms. AMPs have been at the forefront of the response to multidrug-resistant microbial strains and appear to be ideal drug candidates. Lactophoricin (LPcin), naturally produced from bovine milk, is a typical cationic antimicrobial peptide. Three analog peptides, including LPcin-YK5, LPcin-YK8, and LPcin-YK11, with enhanced antimicrobial activity compared to the wild-type LPcin, were designed and expressed in our laboratory. We investigated the structure and antimicrobial mechanisms of action of the three novel antimicrobial peptide analogs derived from LPcin using solution NMR and solid-state NMR spectroscopy in membrane environments. Our results revealed that the three LPcin analogs exhibited helical structures with different tilt angles on the phospholipid membrane surface. We proposed three-dimensional conformations and antibacterial mechanisms of action of the three peptide analogs in the phospholipid bilayers using two-dimensional solid-state separated local field NMR experiments.

Suppressive Effects of a Truncated Inhibitor K562 Protein-Derived Peptide on Two Proinflammatory Cytokines, IL-17 and TNF-α.

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562 cells, a leukemia cell line, and is an inhibitory regulator of interferon-γ-induced major histocompatibility complex class II expression. Recently, exogenous truncated IK (tIK) protein showed potential as a therapeutic agent for inflammation-related diseases. In this study, we designed a novel putative anti-inflammatory peptide derived from tIK protein based on homology modeling of the human interleukin-10 (hIL-10) structure, and investigated whether the peptide exerted inhibitory effects against proinflammatory cytokines such as IL-17 and tumor necrosis factor-α (TNF-α). The peptide contains key residues involved in binding hIL-10 to the IL-10 receptor, and exerted strong inhibitory effects on IL- 17 (43.8%) and TNF-α (50.7%). In addition, we used circular dichroism spectroscopy to confirm that the peptide is usually present in a random coil configuration in aqueous solution. In terms of toxicity, the peptide was found to be biologically safe. The mechanisms by which the short peptide derived from human tIK protein exerts inhibitory effects against IL-17 and TNF-α should be explored further. We also evaluated the feasibility of using this novel peptide in skincare products.

Homology Modeling and Optimized Expression of Truncated IK Protein, tIK, as an Anti-Inflammatory Peptide.

Rheumatoid arthritis, caused by abnormalities in the autoimmune system, affects about 1% of the population. Rheumatoid arthritis does not yet have a proper treatment, and current treatment has various side effects. Therefore, there is a need for a therapeutic agent that can effectively treat rheumatoid arthritis without side effects. Recently, research on pharmaceutical drugs based on peptides has been actively conducted to reduce negative effects. Because peptide drugs are bio-friendly and bio-specific, they are characterized by no side effects. Truncated-IK (tIK) protein, a fragment of IK protein, has anti-inflammatory effects, including anti-rheumatoid arthritis activity. This study focused on the fact that tIK protein phosphorylates the interleukin 10 receptor. Through homology modeling with interleukin 10, short tIK epitopes were proposed to find the essential region of the sequence for anti-inflammatory activity. TH17 differentiation experiments were also performed with the proposed epitope. A peptide composed of 18 amino acids with an anti-inflammatory effect was named tIK-18mer. Additionally, a tIK 9-mer and a 14-mer were also found. The procedure for the experimental expression of the proposed tIK series (9-mer, 14-mer, and 18-mer) using bacterial strain is discussed.

Insight into the bovine milk peptide LPcin-YK3 selection in the proteolytic system of Lactobacillus species.

Antimicrobial peptides are class of small, positively charged peptides known for their broad-spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin-YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100-fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide-binding protein A and LPcin-YK3 peptide. Given that degradation related to the LPcin-YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin-YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide-binding protein A in respect of LPcin-YK3 peptide.

Antimicrobial Activity of Antimicrobial Peptide LPcin-YK3 Derived from Bovine Lactophoricin.

We previously reported on lactophoricin (LPcin), a cationic α-helical antimicrobial peptide derived from bovine milk, which has antimicrobial effects on Candida albicans as well as Gram-positive and Gram-negative bacteria. In this study, we designed the LPcin-YK3 peptide, a shorter analog of LPcin, and investigated its antimicrobial activity. This peptide, consisting of 15 amino acids with + 3 net charges, was an effective antimicrobial agent against the on the Gram-positive strain, Staphylococcus aureus (MIC: 0.62 µg/ml). In addition, the hemolytic activity assay revealed that the peptide was not toxic to mouse and human erythrocytes up to 40 µg/ml. We also used circular dichroism spectroscopy to confirm that peptide in the presence of lipid has α-helical structures and later provide an overview of the relationship between each structure and antimicrobial activity. This peptide is a member of a new class of antimicrobial agents that could potentially overcome the problem of bacterial resistance caused by overuse of conventional antibiotics. Therefore, it could be used as a therapeutic or natural additive, particularly in the cosmetics industry.

Design and Engineering of Antimicrobial Peptides Based on LPcin-YK3, an Antimicrobial Peptide Derivative from Bovine Milk.

We have previously derived a novel antimicrobial peptide, LPcin-YK3(YK3), based on lactophoricin and have successfully studied and reported on the relationship between its structure and function. In this study, antimicrobial peptides with improved antimicrobial activity, less cytotoxicity, and shorter length were devised and characterized on the basis of YK3, and named YK5, YK8, and YK11. The peptide design was based on a variety of knowledge, and a total of nine analog peptides consisted of one to three amino acid substitutions and C-terminal deletions. In detail, tryptophan substitution improved the membrane perturbation, lysine substitution increased the net charge, and excessive amphipathicity decreased. The analog peptides were examined for structural characteristics through spectroscopic analytical techniques, and antimicrobial susceptibility tests were used to confirm their activity and safety. We expect that these studies will provide a platform for systematic engineering of new antibiotic peptides and generate libraries of various antibiotic peptides.

Design, Characterization, and Antimicrobial Activity of a Novel Antimicrobial Peptide Derived from Bovine Lactophoricin.

Lactophoricin (LPcin), which is a part of proteose peptone isolated from bovine milk, is a cationic amphipathic α-helical antimicrobial peptide. Its truncated variants and mutated analogs were designed and their antimicrobial activities were evaluated by using various assays, like broth dilution methods and disk diffusion methods as well as hemolysis assay. Three analogs, LPcin-C8 (LPcin-YK1), LPcin-T2&6W (LPcin-YK2), and LPcin-T2&6W-C8 (LPcin-YK3), which showed better antibiotic activities than LPcin, were selected. Their secondary structures were also characterized by using CD spectropolarimetry. These three analogs of LPcin could be used as an alternative source of powerful antibacterial agents.

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs.

Lactophoricin (LPcin), a component of proteose peptone (113-135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing (1)H-(15)N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting (15)N 1D and 2D (1)H-(15)N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built (1)H-(15)N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55-75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.

Solution and solid-state NMR structural studies of antimicrobial peptides LPcin-I and LPcin-II.

Lactophoricin (LPcin-I) is an antimicrobial, amphiphatic, cationic peptide with 23-amino acid residues isolated from bovine milk. Its analogous peptide, LPcin-II, lacks six N-terminal amino acids compared to LPcin-I. Interestingly, LPcin-II does not display any antimicrobial activity, whereas LPcin-I inhibits the growth of both Gram-negative and Gram-positive bacteria without exhibiting any hemolytic activity. Uniformly (15)N-labeled LPcin peptides were prepared by the recombinant expression of fusion proteins in Escherichia coli, and their properties were characterized by electrospray ionization mass spectrometry, circular dichroism spectroscopy, and antimicrobial activity tests. To understand the structure-activity relationship of these two peptides, they were studied in model membrane environments by a combination of solution and solid-state NMR spectroscopy. We determined the tertiary structure of LPcin-I and LPcin-II in the presence of dodecylphosphorylcholine micelles by solution NMR spectroscopy. Magnetically aligned unflipped bicelle samples were used to investigate the structure and topology of LPcin-I and LPcin-II by solid-state NMR spectroscopy.

Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli.

Lactophoricin (LPcin-I) is a 23-amino acid peptide that corresponds to the carboxyterminal 113-135 region of component-3 of proteose peptone (PP3), a minor phosphoglycoprotein found in bovine milk. It has been reported that lactophoricin has antibacterial activity and a cationic amphipathic helical structure, but its shorter analogous peptide (LPcin-II), a 17-amino acid peptide, corresponding to the 119-135 region of PP3 does not display antibacterial activity. LPcin-I and LPcin-II have similar charge ratios and identical hydrophobic/hydrophilic sectors, according to their helical wheel projection patterns, and both peptides show cationic amphipathic helical folding and interact with membranes. However, it is known that only LPcin-I incorporates into planar lipidic bilayers to form voltage-dependent channels. In this study, the authors cloned and expressed the two recombinant peptides as ketosteroid isomerase (KSI) fusion proteins inclusion bodies in Escherichia coli. These peptides were subjected to NMR structural studies to explore their structure-activity relationships. Fusion proteins were purified by Ni-NTA affinity chromatography under denaturing conditions, and recombinant LPcin-I and LPcin-II were released from fusion by CNBr cleavage. Final purifications of LPcin-I and LPcin-II were achieved by preparative reversed-phase high performance liquid chromatography. Using these methods, we obtained several tens of milligrams of uniformly and selectively (15)N labeled peptides per liter of growth, which was sufficient for solid-state NMR spectroscopy. Peptides were identified by tris-tricine polyacrylamide gel electrophoresis and HSQC spectra. Initial structural data were obtained by solution NMR spectroscopy and compared in membrane-like environments.

High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies.

It has been demonstrated that human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and heterozygous mutations in the hMC4R gene are the most frequent genetic cause of severe human obesity. In order to obtain additional insight into the structure and function, we cloned, expressed, and purified the second transmembrane domain of the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2). To facilitate structural studies of these hMC4R by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled protein are necessary. However, large-scale production of most transmembrane proteins has been limited by experimental adversities due to insufficient yields and low solubility of protein. Nevertheless, through the optimization of the expression and purification approach, we could obtain uniformly or selectively labeled fusion proteins in yields as high as 200-250 mg per liter M9 minimal medium. These proteins were overexpressed in inclusion bodies as a fusion protein with ketosteroid isomerase (KSI) in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography under denaturing conditions. wt-/m-TM2 peptides were released from the fusion by cyanogen bromide cleavage at the Met residue and separated from the carrier KSI by size exclusion chromatography. Initial structural data obtained by solution NMR measurements of wt-/m-TM2 is also presented. The successful application to the production of the second transmembrane domain of human MC4R indicates that the method can be applied to other transmembrane proteins as well and also enable its structural and functional studies using solid-state NMR spectroscopy.

Solid-state NMR spectroscopy of silicon-treated rice with enhanced host resistance against blast.

Silicon is the second-most abundant element on the surface of the earth, and has been considered important for plant growth and development. As for its role in enhanced plant disease resistance, silicon has been reported to reinforce the physical barrier against the penetration and colonization of pathogens. Rice leaves of silicon-treated plants and control plants at the eight- and twelve-leaf growth stages were analyzed by 29Si solid-state nuclear magnetic resonance spectroscopy to characterize the silicon-induced, cell wall fortification of rice leaves, which demonstrated an ability to counter a pathogen attack.

NMR studies of the prionogenic peptide derived from Sup35 protein.

The NMR studies of the prionogenic peptide derived from Sup35 are presented. The peptide molecules were dissolved in the half-aqueous solution to prevent severe aggregation, and were found to be in an extended conformation from the chemical shift and the coupling constant data. They could form higher order multimers by making intermolecular hydrogen bonds, judging from the observation that the NMR sample became a gel-like state at lower temperatures. This work reports the first structural information in the solution state about the prionogenic peptide mimicking the state of amyloid fibrils, and provides a solid foundation for further structural analysis of peptide molecules forming insoluble aggregates.