Molecular investigation of the tandem Tudor domain and plant homeodomain histone binding domains of the epigenetic regulator UHRF2.

Ubiquitin-like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD-PHD domains and compared the binding activity to its UHRF1 counterpart. We found that unlike UHRF1 where the PHD is the primary binding contributor, the TTD of UHRF2 has modestly higher affinity toward the H3 tail, while the PHD has a weaker binding interaction. We also demonstrated that like UHRF1, the aromatic amino acids within the TTD are important for binding to H3K9me3 and a conserved aspartic acid within the PHD forms an ionic interaction with R2 of H3. However, while the aromatic amino acids in the TTD of UHRF1 contribute to selectivity, the analogous residues in UHRF2 contribute to both selectivity and affinity. We also discovered that the PHD of UHRF2 contains a distinct asparagine in the H3R2 binding pocket that lowers the binding affinity of the PHD by reducing a potential electrostatic interaction with the H3 tail. Furthermore, we demonstrate the PHD and TTD of UHRF2 cooperate to interact with the H3 tail and that dual domain engagement with the H3 tail relies on specific amino acids. Lastly, our data indicate that the unique stretch region in the TTD of UHRF2 can decrease the melting temperature of the TTD-PHD and represents a disordered region. Thus, these subtle but important mechanistic differences are potential avenues for selectively targeting the histone binding interactions of UHRF1 and UHRF2 with small molecules.

Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

Using Small Peptide Segments of Amyloid-ß and Humanin to Examine their Physical Interactions.

BACKGROUND: Amyloid fibrils in Alzheimer's disease are composed of amyloid-ß (Aß) peptides of variant lengths. Humanin (HN), a 24 amino acid residue neuroprotective peptide, is known to interact with the predominant Aß isoform in the brain, Aß (1-40). METHODS: Here, we constructed smaller segments of Aß and HN and identified residues in HN important for both HN-HN and HN-Aß interactions. Peptides corresponding to amino acid residues 5- 15 of HN, HN (5-15), HN (5-15, L11S), where Leu11 was replaced with Ser, and residues 17-28 of Aß, Aß (17-28), were synthesized and tested for their ability to block formation of the complex between HN and Aß (1-40). RESULTS: Co-immunoprecipitation and binding kinetics showed that HN (5-15) was more efficient at blocking the complex between HN and Aß (1-40) than either HN (5-15, L11S) or Aß (17-28). Binding kinetics of these smaller peptides with either full-length HN or Aß (1-40) showed that HN (5- 15) was able to bind either Aß (1-40) or HN more efficiently than HN (5-15, L11S) or Aß (17-28). Compared to full-length HN, however, HN (5-15) bound Aß (1-40) with a weaker affinity suggesting that while HN (5-15) binds Aß, other residues in the full length HN peptide are necessary for maximum interactions. CONCLUSION: L11 was more important for interactions with Aß (1-40) than with HN. Aß (17-28) was relatively ineffective at binding to either Aß (1-40) or HN. Moreover, HN, and the smaller HN (5-15), HN (5-15 L11S), and Aß (17-28) peptides, had different effects on regulating Aß (1-40) aggregation kinetics.

Characterizing the binding of dopamine D1-D2 receptors in vitro and in temporal and frontal lobe tissue total protein.

Dysfunction of the dopaminergic pathway is linked to numerous diseases of the nervous system. The D1-D2 receptor heteromer is known to play a role in certain neuropsychiatric disorders, such as depression. Here, we synthesized an eight amino acid residue peptide, EAARRAQE, derived from the third intracellular loop of the D2 receptor and show that the peptide binds to the D1 receptor with comparable efficiency as that of the full-length D2 receptor protein. Moreover, immunoprecipitation studies show the existence of a heteromeric complex formed both in vitro and in total protein derived from temporal and frontal lobe tissue from normal and depressed subjects. The efficiency of the peptide to block the D1-D2 heteromeric complex was comparable in all the samples tested.

D-Amino Acid Analogues of the Antimicrobial Peptide CDT Exhibit Anti- Cancer Properties in A549, a Human Lung Adenocarcinoma Cell Line.

INTRODUCTION: The importance of the antitumor activity of some antimicrobial peptides (AMPs) is being increasingly recognized. The antimicrobial peptide, tachyplesin, has been shown to exhibit anticancer properties and a linear, cysteine deleted analogue (CDT), was found to retain its antibacterial function. OBJECTIVES: The objective was to test CDT and related analogues against normal mammalian, bacterial, and cancer cells to determine their effectiveness and then utilize specific assays to determine a possible mechanism of action. METHODS: We used sequence reversal and D-amino acids to synthesize four CDT analogues by solid phase peptide synthesis. A number of assays were used including liposome dye-leakage, antibacterial activity against both Gram-positive and Gram-negative bacterial strains, hemolytic assays, methyl thiazolyl tetrazolium (MTT), and apoptosis to examine their effectiveness as both AMPs and anti-cancer peptides (ACPs). We then tested the analogues for their ability to inhibit proliferation of the human lung cancer cell line, A549. RESULTS: We found that D-CDT exhibited the best bactericidal properties of those tested and was not damaging to red blood cells. Both D-CDT and reverse D-CDT showed a dose-dependent reduction of cell viability. However, D-CDT was most effective with an IC50 of 9.814 µM, a value 9-fold lower than that calculated for reverse D-CDT (90.16 µM). Apoptosis does not appear to be a mechanism by which D-CDT exerts its anticancer properties since > 100 µM was required to increase activation of caspase 3. Moreover, the ERK1/2 pathway is also unlikely since only a modest (20%) decrease of activity was observed with > 100 µM D-CDT. However, D-CDT was found to operate via a hyaluronan (HA)-dependent mechanism as pretreatment of the cells with hyaluronidase decreased the cytotoxic effects of D-CDT on A549 cells and increased its IC50 29-fold to 283.9 µM. CONCLUSION: D-CDT is both an effective AMP and ACP, and likely exerts its anticancer effects through both membranolytic as well as an HA-mediated mechanism.

Humanin Peptide Binds to Insulin-Like Growth Factor-Binding Protein 3 (IGFBP3) and Regulates Its Interaction with Importin-ß.

Nuclear translocation of IGFBP3 by importin-ß1 is a prerequisite for IGFBP3-induced apoptosis. The neuroprotective peptide humanin (HN) counteracts IGFBP3-induced cell death. However, the mechanism by which humanin protects cells is currently unknown. The natural synthesis of this peptide decreases with age, coincident with the likelihood for the development of Alzheimer's Disease, making it a promising target for therapeutics. We have examined the effect of full-length humanin and a synthetic analogue (HN 3-19), known to be sufficient for its neuroprotective function, on the interaction between IGFBP3 and importin-ß1. Using competitive ligand dot blotting, co-immunoprecipitation, and an ELISA-based binding assay, we determined that 1) humanin binds to IGFBP3 with a Kd of 5.05 µM and 2) both humanin (IC50 of 18.1 µM) and HN 3-19 (IC50 of 10.3 µM) interfere with the binding of importin-ß1 to IGFBP3 in vitro. We also demonstrated that HN 3-19 is able to reduce the rate of apoptosis in a human lung adenocarcinoma cell line, suggesting a possible mechanism of action for humanin as an inhibitor of IGFBP3 nuclear translocation. Understanding the exact mechanism by which humanin and its analogue, HN 3-19, bind to IGFPB3 and regulate its interaction with importin-ß1 will open the door to modulating the protein-protein interactions involved in neuronal cell death.

Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: studies of IAPP(20-29) fragments.

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.

Modeling the interface between islet amyloid polypeptide and insulin-based aggregation inhibitors: correlation to aggregation kinetics and membrane damage.

Human islet amyloid polypeptide (hIAPP) forms cytotoxic fibrils in type-2 diabetes and insulin is known to inhibit formation of these aggregates. In this study, a series of insulin-based inhibitors were synthesized and assessed for their ability to slow aggregation and impact hIAPP-induced membrane damage. Computational studies were employed to examine the underlying mechanism of inhibition. Overall, all compounds were able to slow aggregation at sufficiently high concentrations (10× molar excess); however, only two peptides showed any inhibitory capability at the 1:1 molar ratio (EALYLV and VEALYLV). The results of density functional calculations suggest this is due to the strength of a salt bridge formed with the Arg11 side chain of hIAPP and the inhibitors' ability to span from the Arg11 to past the Phe15 residue of hIAPP, blocking one of the principal amyloidogenic regions of the molecule. Unexpectedly, slowing fibrillogenesis actually increased damage to lipid membranes, suggesting that the aggregation process itself, rather than the fibrilized peptide, may be the cause of cytotoxicity in vivo.

Antimicrobial and membrane disrupting activities of a peptide derived from the human cathelicidin antimicrobial peptide LL37.

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an alpha-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. (15)N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. (31)P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

Correlation of LUMO localization with the alpha-amylase inhibition constant in a Tendamistat-based series of linear and cyclic peptides.

The glycosidase alpha-amylase is responsible for the hydrolysis of alpha(1-->4) glycosidic linkages found in dietary starch as one means for controlling blood sugar level. The effect of alpha-amylase is detrimental, however, in the disease state diabetes mellitus, where blood glucose levels are elevated due to a biochemical defect. Inhibition of the enzyme's activity would reduce glucose absorption by the small intestine. Our objective was to develop small peptides based on essential binding elements of the natural protein inhibitor, Tendamistat. These smaller analogs may be better studied structurally and conformationally to help us understand molecular-level interactions. In addition, we have been able to correlate the activity of our compounds with the lowest unoccupied molecular orbital (LUMO) localization in energy-minimized conformations. The positive charge/LUMO of most active inhibitors is localized on the central Arg residue of the required triplet. This provides a predictive model for the design of active molecules.

Peptide inhibitors of alpha-amylase based on tendamistat: development of analogues with omega-amino acids linking critical binding segments.

Peptide analogues of Tendamistat which include the most essential residues linked by novel omega-amino acids (X,Y,Z: H2N-(CH2)n-CO(2)H, where n=2-10) were designed, synthesized (Ac-Tyr(15)-X-Trp(18)-Arg(19)-Tyr(20)-Y-Thr(55)-Z-Asp(58)-Gly(59)-Tyr(60)-Ile(61)-Gly(62)-NH2), and analyzed for alpha-amylase inhibitory activity. Native dipeptide spacers sometimes were left intact at X and Z. Analogues demonstrated competitive inhibition with K(i) values ranging from 23 to 767 microM. 8-Aminooctanoic acid was the optimal linker at Y, while longer linkers were favored at the other positions.

pKa and volume of residue one influence delta/mu opioid binding: QSAR analysis of tyrosine replacement in a nonselective deltorphin analogue.

[Gly(4)]deltorphin (Tyr-D-Ala-Phe-Gly-Val-Val-Gly-NH(2)) is a nonselective analogue of the opioid heptapeptides isolated from Phyllomedusa amphibian skin. Its nonselective nature allows for simultaneous characterization of the effects of sequence modification on both delta (delta) and mu (mu) receptor binding. The N-terminal regions of opioid peptides are considered to be responsible for receptor recognition, and the tyrosine at position one is relatively intolerant to alteration. In order to further investigate the role of the phenolic hydroxyl group in receptor interaction, a series of peptides was synthesized in which the position-one tyrosine residue was replaced with analogues of varying electronic, steric, and acid/base character, including ring-substituted tyrosines, para-substituted phenylalanines, and other nonaromatic and heterocyclic amino acids. The effects of these replacements on delta and mu receptor affinities were measured and then analyzed through quantitative structure-activity relationship (QSAR) calculations. Results support a dual hydrogen bond donor/acceptor role for the Tyr(1) hydroxyl moiety, with less acidic hydroxyl groups exhibiting stronger binding to opioid receptors. In addition, steric bulk in the Tyr(1) position independently strengthens mu and possibly delta binding, presumably by either a ligand conformational effect or enhanced van der Waals interactions with a 'loose' receptor site. The pK(a) effect is stronger on delta than on mu binding, generating an increase in delta selectivity with increasing residue-one pK(a).