Stable Polyglutamine Dimers Can Contain β-Hairpins with Interdigitated Side Chains—But Not α-Helices, β-Nanotubes, β-Pseudohelices,or Steric Zippers

A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation.     

Permeation of a β-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS-(S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)-β³hAla-(S)-β³hArg-(R)-β³hLeu-(S)-β³hPhe-(S)-β³hAla-(S)-β³hAla-(S)-β³hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb³⁺-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Effect of 6α,7β-dihydroxyvouacapan-17β-oic acid and its lactone derivatives on the growth of human cancer cells

The furanditerpene 6α,7β-dihydroxyvouacapan-17β-oic acid (1) is a natural product biosynthesized by some species from the genus Pterodon (Leguminosae). This secondary metabolite has multiple biological activities that include anti-inflammatory, analgesic, plant growth regulatory, anti-edematogenic, photosystem II inhibitory and photosynthesis uncoupler, and antifungal properties. However, few studies on the antiproliferative profile of compound 1 and/or its derivatives have been reported up to date. Here, we describe the isolation of compound 1 from hexane extract of P. polygalaeflorus fruits as well as the semisynthesis of three lactone derivatives: 6α-hydroxyvouacapan-7β,17β-lactone (2), 6α-acetoxyvouacapan-7β,17β-lactone (3), and 6-oxovouacapan-7β,17β-lactone (4). Additionally, antiproliferative activity of these compounds against nine human cancer cell lines was investigated. Our results revealed that 6α-hydroxyvouacapan-7β,17β-lactone (2) was the most potent furanditerpene against all cancer cell lines studied. The presence of non-substituted hydroxyl group at C-6 and the presence of 7β,17β-lactone ring are important for the antiproliferative activity of these compounds.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Hb Villaverde [β 89 (F5) Ser → Thr]: the structural modification of an intrasubunit contact is responsible for a high oxygen affinity

Hb Villaverde [β89 (F5) Ser → Thr], identified in a Spanish patient, is a new human hemoglobin variant, electrophoretically silent, responsible for a severe erythrocytosis. This abnormal hemoglobin displays a very high oxygen affinity and a markedly reduced cooperativity that is partly restored in the presence of IHP. Determination of the structural abnormality was achieved on a mixture of the normal and abnormal β-chains. After isolation of the abnormal tryptic peptide by RP-HPLC, its sequence was determined by mass spectrometry. The structural abnormality disturbs the intrasubunit interaction between helices F and H and, thus, may weaken the C-terminal bonds of the deoxy conformation and the heme contacts of several hydrophobic residues. Hb Villaverbe demonstrates that this intrasubunit contact between helices F and H is essential for the cohesion of the hemoglobin molecule.     

Kaiso is a bimodal modulator for Wnt/β-catenin signaling

The Wnt family of secreted ligands plays critical roles during embryonic development and tumorigenesis. Here we show that Kaiso, a dual specific DNA-binding protein, functions as a bimodal regulator of canonical Wnt signaling. Loss-of-function analysis of Kaiso abrogated Wnt-mediated reporter activity and axis duplication, whereas gain-of-function analysis of Kaiso dose-dependently resulted in synergistic and suppressive effects. Our analyses further suggest Kaiso can regulate TCF/LEF1-activity for these effects via modulating HDAC1 and β-catenin-complex formation. Our studies together provide insights into why Kaiso null mice display resistance to intestinal tumors when crossed onto an Apc^Min/+ background.

Stuctured summary

MINT-6823807: HDAC1 (uniprotkb:Q13547) physically interacts (MI:0218) with beta catenin (uniprotkb:P35222) by anti tag coimmunoprecipitation (MI:0007)MINT-6823820: axin (uniprotkb:O15169) physically interacts (MI:0218) with beta catenin (uniprotkb:P35222) by anti tag coimmunoprecipitation (MI:0007)     

Aggregation Modulators Interfere with Membrane Interactions of β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β₂-microglobulin (β₂m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β₂m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.     

hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein codinghsrω locus inDrosophila melanogaster

Thehrsω or the 93D heat shock locus ofDrosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz.,Df(3R) e ^Gp4 (eGp4) andDf(3R)GC14 (GC14), is known to cause a high (∼ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of thehsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of thehsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when onehsp83 mutant allele (hsp83 ^e4A) is introduced ineGp4/GC14 trans-heterozygotes, practically none of thehsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction betweenhsp83 andhsrω genes was further confirmed by examining the effect of thehsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that thehsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for thehsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development ofDrosophila reveals novel aspects of their biological functions.     

Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

Folding Simulations of a De Novo Designed Protein with a βαβ Fold

βαβ structural motifs are commonly used building blocks in protein structures containing parallel β-sheets. However, to our knowledge, no stand-alone βαβ structure has been observed in nature to date. Recently, for the first time that we know of, a small protein with an independent βαβ structure (DS119) was successfully designed in our laboratory. To understand the folding mechanism of DS119, in the study described here, we carried out all-atom molecular dynamics and coarse-grained simulations to investigate its folding pathways and energy landscape. From all-atom simulations, we successfully observed the folding event and got a stable folded structure with a minimal root mean-square deviation of 2.6 Å with respect to the NMR structure. The folding process can be described as a fast collapse phase followed by rapid formation of the central helix, and then slow formation of a parallel β-sheet. By using a native-centric Gō-like model, the cooperativity of the system was characterized in terms of the calorimetric criterion, sigmoidal transitions, conformation distribution shifts, and free-energy profiles. DS119 was found to be an incipient downhill folder that folds more cooperatively than a downhill folder, but less cooperatively than a two-state folder. This may reflect the balance between the two structural elements of DS119: the rapidly formed α-helix and the slowly formed parallel β-sheet. Folding times estimated from both the all-atom simulations and the coarse-grained model were at microsecond level, making DS119 another fast folder. Compared to fast folders reported previously, DS119 is, to the best of our knowledge, the first that exhibits a parallel β-sheet.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

δN and δC analysis of a Posidonia sinuosa seagrass bed

Potential food sources and dominant invertebrates and fishes were collected for the examination of variability in ¹³C/¹²C and ¹⁵N/¹⁴N to determine the sources of carbon available to consumers within a Western Australian Posidonia sinuosa-dominated seagrass bed. Autotrophs showed a wide distribution of δ¹³C values, with P. sinuosa at −11.3 ± 0.8‰ and macroalgae ranging from −16.6 to −31.7‰. This variation allowed us to successfully identify macroalgae as the main contributor of carbon to the trophic structure, although no distinction could be made between epiphytic macroalgae on seagrass, or allochthonous macroalgal sources. The range in δ¹⁵N ratios among potential food items at the trophic base was too small to make it useful as tracer of nitrogen flow pathways, but it consistently increased from macrophytes and detritus (4.1–6.8‰), to invertebrates (5.7–7.4‰) located near the middle of the food web, to fishes (8.3–11.9‰), with piscivorous species such as Leviprora inops generally having a higher ¹⁵N. δ¹³C of seston (−12.8‰) and sedimentary organic matter (−8.7‰) indicate that seagrass material is the main contributor to these two carbon pools, and that very little of it contributes to animal biomass.     

Thermodynamics of β-Sheet Formation in Polyglutamine

The role of β-sheets in the early stages of protein aggregation, specifically amyloid formation, remains unclear. Interpretations of kinetic data have led to a specific model for the role of β-sheets in polyglutamine aggregation. According to this model, monomeric polyglutamine, which is intrinsically disordered, goes through a rare conversion into an ordered, metastable, β-sheeted state that nucleates aggregation. It has also been proposed that the probability of forming the critical nucleus, a specific β-sheet conformation for the monomer, increases with increasing chain length. Here, we test this model using molecular simulations. We quantified free energy profiles in terms of β-content for monomeric polyglutamine as a function of chain length. In accord with estimates from experimental data, the free energy penalties for forming β-rich states are in the 10-20 kcal/mol range. However, the length dependence of these free energy penalties does not mirror interpretations of kinetic data. In addition, although homodimerization of disordered molecules is spontaneous, the imposition of conformational restraints on polyglutamine molecules does not enhance the spontaneity of intermolecular associations. Our data lead to the proposal that β-sheet formation is an attribute of peptide-rich phases such as high molecular weight aggregates rather than monomers or oligomers.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

CR1 — a dispersed repeated element associated with the Cab-1 locus in tomato

Cab-1 is a complex genetic locus in tomato consisting of four clustered genes encoding chlorophyll a/b-binding polypeptide. Southern blot analysis of total tomato DNA with genomic clones corresponding to the Cab-1 locus has revealed the presence of a repetitive element in the 3 kb spacer regions between two of these genes. This repetitive element, named CR1, has been characterized via sequencing, genetic mapping and hybridization to related solanaceous species. Results indicate that there are as many as 30 copies of this element in the tomato genome and that most, if not all, are found at independent loci. Sites corresponding to 12 of the repeats have been located on different regions of chromosomes 2, 4, 5, 7, 10 and 11. A 1.6 kb PstI-EcoRI fragment from the Cab-1 locus containing the element was sequenced and found to be 75% AT-rich. No open reading frames larger than 150 bp were detected. Several imperfect inverted repeats flanked by direct repeats could be found at the ends of the element. This arrangement is reminiscent of known transposons. Southern hybridization analysis indicates that multiple copies of CR1 exist in all species of the genus Lycopersicon as well as in Solanum lycopersicoides and S. tuberosum (potato), but not in eggplant, pepper, petunia, Datura or tobacco. Melt-off experiments indicate that members of the CR1 family in the tomato genome are more closely related to one another than to homologous members in the genomes of S. lycopersicoides or S. tuberosum, suggesting some type of concerted evolution.     

Physical activity is associated with slower epigenetic ageing—Findings from the Rhineland study

Epigenetic ageing, i.e., age-associated changes in DNA methylation patterns, is a sensitive marker of biological ageing, a major determinant of morbidity and functional decline. We examined the association of physical activity with epigenetic ageing and the role of immune function and cardiovascular risk factors in mediating this relation. Moreover, we aimed to identify novel molecular processes underlying the association between physical activity and epigenetic ageing. We analysed cross-sectional data from 3567 eligible participants (mean age: 55.5 years, range: 30–94 years, 54.8% women) of the Rhineland Study, a community-based cohort study in Bonn, Germany. Physical activity components (metabolic equivalent (MET)-Hours, step counts, sedentary, light-intensity and moderate-to-vigorous intensity activities) were recorded with accelerometers. DNA methylation was measured with the Illumina HumanMethylationEPIC BeadChip. Epigenetic age acceleration (Hannum's age, Horvath's age, PhenoAge and GrimAge) was calculated based on published algorithms. The relation between physical activity and epigenetic ageing was examined with multivariable regression, while structural equation modeling was used for mediation analysis. Moreover, we conducted an epigenome-wide association study of physical activity across 850,000 CpG sites. After adjustment for age, sex, season, education, smoking, cell proportions and batch effects, physical activity (step counts, MET-Hours and %time spend in moderate-to-vigorous activities) was non-linearly associated with slower epigenetic ageing, in part through its beneficial effects on immune function and cardiovascular health. Additionally, we identified 12 and 7 CpGs associated with MET-Hours and %time spent in moderate-to-vigorous activities, respectively (p < 1 × 10⁻⁵). Our findings suggest that regular physical activity slows epigenetic ageing by counteracting immunosenescence and lowering cardiovascular risk.     

Antibacterial activity of 3,3′-dihydroxycurcumin (DHC) is associated with membrane perturbation

Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial activity. However, the therapeutic uses of this compound are limited due to its chemical instability. In this work, we evaluated the antimicrobial activity of diphenylheptanoids derived from curcumin against Gram-positive and Gram-negative bacteria, and also against Mycobacterium tuberculosis in terms of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values. 3,3′-Dihydroxycurcumin (DHC) displayed activity against Enterococcus faecalis, Staphylococcus aureus and M. tuberculosis, demonstrating MIC values of 78 and 156 µg/mL. In addition, DHC was more stable than curcumin in acetate buffer (pH 5.0) and phosphate buffer (pH 7.4) for 24 h at 37 °C. We proposed that membrane and the cell division protein FtsZ could be the targets for DHC due to that fact that curcumin exhibits this mode of antibacterial action. Fluorescence microscopy of Bacillus subtilis stained with SYTO9 and propidium iodide fluorophores indicated that DHC has the ability to perturb the bacterial membrane. On the other hand, DHC showed a weak inhibition of the GTPase activity of B. subtilis FtsZ. Toxicity assay using human cells indicated that DHC has moderate capacity to reduce viability of liver cells (HepG2 line) and lung cells (MRC-5 and A549 lines) when compared with doxorubicin. Alkaline comet assay indicated that DHC was not able to induce DNA damage in A549 cell line. These results indicated that DHC is promising compound with antibacterial and antitubercular activities.     

An insertion/deletion polymorphism in the 3′ untranslated region of β-transducin repeat-containing protein (βTrCP) is associated with susceptibility for hepatocellular carcinoma in Chinese

Hepatocellular carcinoma (HCC) is an epithelial cancer which originates from hepatocytes or their progenitors. As a positive regulator of NFκB signaling pathway, β-transducin repeat-containing protein (βTrCP) is overexpressed and oncogenic in epithelial cancers, suggesting a potential role of βTrCP in HCC susceptibility. We carried out a case-control study in a Chinese population (256 cases and 367 controls) to estimate the susceptibility to HCC associated with a 9 bp insertion/deletion polymorphism (rs16405) in 3′ untranslated region of βTrCP. Using unconditional logistic regression, we found that 9N del/del and 9N ins/del genotypes were significantly associated with decreased HCC risk: OR = 0.44 (0.24-0.83) (p = 0.004) and OR = 0.56 (0.31-1.00) (p = 0.034), respectively. Furthermore, in vivo experiments showed that mRNA levels of βTrCP from HCC tumor tissues were correlated with rs16405 genotypes. HCC tumor tissues with homozygous for 9N ins/ins has the highest level of βTrCP, which are 3.99 and 7.04-fold higher than heterozygous 9N ins/del and homozygous 9N del/del, respectively. Based on bioinformatics prediction, we found that the risk allele for rs16405 disrupted a binding site for human microRNA-920 which would negatively regulate βTrCP. We propose a microRNA-920 mediated βTrCP regulation model depending on rs16405 genotype, which warrants further replication association studies and follow-up functional experiments.