Uracil-amino acid as a scaffold for β-sheet peptidomimetics: Study of photophysics and interaction with BSA protein

We report herein the uracil-di-aza-amino acid (U^rAA) as a new family of molecular scaffold to induce β-hairpin structure with H-bonded β-sheet conformation in a short peptide. This has been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine and/or triazolylmethoxynapthyl alanine (^TPyAla_Do and/or ^TMNapAla_Do) are embedded into two arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR, variable temperature and 2D NMR spectroscopy reveals the β-hairpin structures for both the peptides. Study of photophysical property reveals that the pentapeptide containing fluorescent triazolyl unnatural amino acids ^TMNapAla_Do and ^TPyAla_Do at the two termini exhibits dual path entry to exciplex emission-either via FRET from ^TMNapAla_Do to ^TPyAla_Do or via direct excitation of a FRET acceptor, ^TPyAla_Do. The other pentapeptide with ^TPyAla_Do/^TPyAla_Do pair shows excimer emission. Furthermore, both the peptides maintaining their fundamental photophysics are found to interact with BSA as only a test biomolecule.     

A 68Ga complex based on benzofuran scaffold for the detection of β-amyloid plaques

Since the imaging of β-amyloid (Aβ) plaques in the brain is believed to be a useful tool for the early diagnosis of Alzheimer’s disease (AD), a number of imaging probes to detect Aβ plaques have been developed. Because the radionuclide ⁶⁸Ga (t_1/2 = 68 min) for PET imaging could become an attractive alternative to ¹¹C and ¹⁸F, we designed and synthesized a benzofuran derivative conjugated with a ⁶⁸Ga complex (⁶⁸Ga-DOTA-C3-BF) as a novel Aβ imaging probe. In an in vitro binding assay, Ga-DOTA-C3-BF showed high affinity for Aβ(1-42) aggregates (K_i = 10.8 nM). The Ga-DOTA-C3-BF clearly stained Aβ plaques in a section of Tg2576 mouse, reflecting the affinity for Aβ(1-42) aggregates in vitro. In a biodistribution study in normal mice, ⁶⁸Ga-DOTA-C3-BF displayed low initial uptake (0.45% ID/g) in the brain at 2 min post-injection. While improvement of the brain uptake of ⁶⁸Ga complexes appears to be essential, these results suggest that novel PET imaging probes that include ⁶⁸Ga as the radionuclide for PET may be feasible.     

Growing with siblings: a common ground for cooperation or for fiercer competition among plants?

Recent work has shown that certain plants can identify their kin in competitive settings through root recognition, and react by decreasing root growth when competing with relatives. Although this may be a necessary step in kin selection, no clear associated improvement in individual or group fitness has been reported to qualify as such. We designed an experiment to address whether genetic relatedness between neighbouring plants affects individual or group fitness in artificial populations. Seeds of Lupinus angustifolius were sown in groups of siblings, groups of different genotypes from the same population and groups of genotypes from different populations. Both plants surrounded by siblings and by genotypes from the same population had lower individual fitness and produced fewer flowers and less vegetative biomass as a group. We conclude that genetic relatedness entails decreased individual and group fitness in L. angustifolius. This, together with earlier work, precludes the generalization that kin recognition may act as a widespread, major microevolutionary mechanism in plants.     

Toward a common aggregation mechanism for a β-barrel protein family: Insights derived from a stable dimeric species

Δ78Δ is a second generation functional all-β sheet variant of IFABP (intestinal fatty acid binding protein) corresponding to the fragment 29–106 of the parent protein. This protein and its predecessor, Δ98Δ (segment 29–126 of IFABP), were initially uncovered by controlled proteolysis. Remarkably, although IFABP and Δ98Δ are monomers in solution, Δ78Δ adopts a stable dimeric structure. With the aim of identifying key structural features that modulate the aggregation of β-proteins, we evaluate here the structure and aggregation propensity of Δ78Δ. The 2,2,2-trifluoroethanol (TFE) induced aggregation of this protein shows a primary nucleation–elongation mechanism, characterized by the stabilization of a dimeric nucleus. Its rate of production from the co-solvent induced aggregation prone state governs the kinetics of polymerization. In this context, the value of Δ78Δ lies in the fact that – being a stable dimeric species – it reduces an otherwise bimolecular reaction to a unimolecular one. Interestingly, even though Δ78Δ and IFABP display similar conformational stability, the abrogated form of IFABP shows an enhanced aggregation rate, revealing the ancillary role played on this process by the free energy of the native proteins. Δ78Δ share with IFABP and Δ98Δ a common putative aggregation-prone central peptide. Differences in the exposure/accessibility of this segment dictated by the environment around this region might underlie the observed variations in the speed of aggregation. Lessons learnt from this natural dimeric protein might shed light on the early conformational events leading to β-conversion from barrels to amyloid aggregates.     

Looking for the minimum common denominator in haem–copper oxygen reductases: Towards a unified catalytic mechanism

Haem–copper oxygen reductases are transmembrane protein complexes that reduce dioxygen to water and pump protons across the mitochondrial or periplasmatic membrane, contributing to the transmembrane difference of electrochemical potential. Seven years ago we proposed a classification of these enzymes into three different families (A, B and C), based on the amino acid residues of their proton channels and amino acid sequence comparison, later supported by the so far identified characteristics of the catalytic centre of members from each family. The three families have in common the same general structural fold of the catalytic subunit, which contains the same or analogous prosthetic groups, and proton channels. These observations raise the hypothesis that the mechanisms for dioxygen reduction, proton pumping and the coupling of the two processes may be the same for all these enzymes. Under this hypothesis, they should be performed and controlled by the same or equivalent elements/events, and the identification of retained elements in all families will reveal their importance and may prompt the definition of the enzyme operating mode. Thus, we believe that the search for a minimum common denominator has a crucial importance, and in this article we highlight what is already established for the haem–copper oxygen reductases and emphasize the main questions still unanswered in a comprehensive basis.     

A β-tryptase inhibitor with a tropanylamide scaffold to improve in vitro stability and to lower hERG channel binding affinity

Tropanylamide was investigated as a possible scaffold for β-tryptase inhibitors with a basic benzylamine P1 group and a substituted thiophene P4 group. Comparing to piperidinylamide, the tropanylamide scaffold is much more rigid, which presents less opportunity for the inhibitor to bind with off-target proteins, such as cytochrome P450, SSAO, and hERG potassium channel. The proposed binding mode was further confirmed by an in-house X-ray structure through co-crystallization.     

Case for an RNA–prion world: a hypothesis based on conformational diversity

Prions and other misfolded proteins can impart their structure and functions to normal molecules. Based upon a thorough structural assessment of RNA, prions and misfolded proteins, especially from the perspective of conformational diversity, we propose a case for co-existence of these in the pre-biotic world. Analyzing the evolution of physical aspects of biochemical structures, we put forward a case for an RNA–prion pre-biotic world, instead of, merely, the “RNA World”.     

A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine

tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2′,3′-cyclic phosphate and 5′-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2′-PO₄ specificity by showing that AtRNL is deficient in transferring AMP to pRNA_OH to form AppRNA_OH but proficient at sealing pre-adenylylated AppRNA_OH. This strategy for discriminating 2′-PO₄ versus 2′-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific “capping” of 5′-PO₄ ends.     

Atrial fibrillation in β-thalassemia patients with a focus on the role of iron-overload and oxidative stress: A review

Cardiac complications including arrhythmia and especially atrial fibrillation (AF) are common causes of death in β-thalassemia patients. The main factor in the etiopathogenesis of these complications is iron overload, which results in increased oxidative stress. Although there is a known association between cardiac complications and iron overload in β-thalassemia patients, there is no comprehensive review on AF and excessive iron with a focus on oxidative stress in these patients. The aim of this article was to review the different aspects of AF in β-thalassemia patients with a focus on the prevention and treatment of AF by using iron chelators and/or anti-oxidants. AF in β-thalassemia patients is more common than in the general population. One of the most important causes of AF is cardiac iron overload and the harmful effects of increased oxidative stress. Iron-induced AF can be reversed by using an intensive iron chelation regimen. Based on a few experimental studies, the combination of iron chelators with some anti-oxidants, including NAC, vitamin C, and acetaminophen, can lead to improved cardiac protection. However, the effect of such combinations on cardiac arrhythmias should be further evaluated with animal and human studies.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Connexin43 interacts with βarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43-interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.     

A second catalytic domain in the Elp3 histone acetyltransferases: a candidate for histone demethylase activity?

A new subfamily of two-domain histone acetyltransferases (HATs) related to Elp3 has been identified. In addition to a HAT domain in the C terminus, these proteins have an N-terminal domain similar to the catalytic domain of S-adenosylmethionine radical enzymes. Two-domain organization is preserved in evolution, suggesting that both enzymatic activities are functionally or mechanistically coupled and directed towards highly conserved substrates. The functional implications of this similarity and a possible role for Elp3-related proteins as histone demethylases are discussed.     

Effect of zinc binding on β-amyloid structure and dynamics: implications for Aβ aggregation

Assembly of β-amyloid (Aβ) peptide into toxic oligomers is widely believed to initiate Alzheimer's disease pathogenesis. Under in vitro physiological conditions, zinc (Zn(II)) can bind to Aβ and redirect its assembly from amyloid fibrillar toward less toxic amorphous aggregation. Propensity of Aβ to go toward a specific form of aggregate state is determined by structural and dynamical properties of the initial monomeric as well as the aggregate state. Here we probe the structural and dynamical impact of binding of Zn(II) to monomeric Aβ40 using NMR spectroscopy. To obtain further support for the importance of intrinsic dynamics in the aggregation precursor, ¹⁵N relaxation measurements were also performed for Aβ42, the more fibrillar aggregation-prone variant of Aβ. The combined data suggest that, upon Zn(II)-binding to the N-terminus of Aβ40, a relatively rigid turnlike structure is induced at residues Val²⁴-Lys²⁸ whereas the residues flanking this region become more mobile on the picosecond-to-nanosecond timescale. This is in contrast to the increased rigidity of Aβ42 at the C-terminus, and proposed to be linked to the higher propensity of Zn(II)-bound peptide to form amorphous aggregates with less entropic penalties than their fibrillar counterparts.     

RNA–LIM: A novel procedure for analyzing protein/single-stranded RNA propensity data with concomitant estimation of interface structure

RNA–LIM is a procedure that can analyze various pseudo-potentials describing the affinity between single-stranded RNA (ssRNA) ribonucleotides and surface amino acids to produce a coarse-grained estimate of the structure of the ssRNA at the protein interface. The search algorithm works by evolving an ssRNA chain, of known sequence, as a series of walks between fixed sites on a protein surface. Optimal routes are found by application of a set of minimal “limiting” restraints derived jointly from (i) selective sampling of the ribonucleotide amino acid affinity pseudo-potential data, (ii) limited surface path exploration by prior determination of surface arc lengths, and (iii) RNA structural specification obtained from a statistical potential gathered from a library of experimentally determined ssRNA structures. We describe the general approach using a NAST (Nucleic Acid Simulation Tool)-like approximation of the ssRNA chain and a generalized pseudo-potential reflecting the location of nucleic acid binding residues. Minimum and maximum performance indicators of the methodology are established using both synthetic data, for which the pseudo-potential defining nucleic acid binding affinity is systematically degraded, and a representative real case, where the RNA binding sites are predicted by the amplified antisense RNA (aaRNA) method. Some potential uses and extensions of the routine are discussed. RNA–LIM analysis programs along with detailed instructions for their use are available on request from the authors.     

Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

-Keto esters derived from dipeptides areprepared by application of common methodologiesemployed for the synthesis of amino acid-derived-keto esters; however, epimerization of theC-terminal residue occurred to different extentsdepending on the method. In imidazolide activateddipeptides, this epimerization is due to the CDIactivation step and to the configurational instabilityof the intermediate imidazolides in different reactionmedia. Regarding yield and diastereomeric purity, themethod of choice proved to be the reaction ofdipeptide-derived imidazolide with the potassium saltof malonic half esters in the presence of MgCl₂.     

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid-base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.     

Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

Summary β-Keto esters derived from dipeptides are prepared by application of common methodologies employed for the synthesis of amino acid-derived β-keto esters; however, epimerization of the C-terminal residue occurred to different extents depending on the method. In imidazolide activated dipeptides, this epimerization is due to the CDI activation step and to the configurational instability of the intermediate imidazolides in different reaction media. Regarding yield and diastereomeric purity, the method of choice proved to be the reaction of dipeptide-derived imidazolide with the potassium salt of malonic half esters in the presence of MgCl₂.     

A sialidase complex from chicken liver: Characterization of a multienzyme complex with β-galactosidase and carboxypeptidase

Mammalian lysosomal sialidase exists as an enzyme complex with β-galactosidase and carboxypeptidase, so-called “protective protein.” In this article, we report that chicken sialidase also occurs as a complex with β-galactosidase and protective protein. The purified sialidase complex had a molecular weight > 700 kDa on gel filtration and showed four protein components of 76, 65, 54 and 48 kDa on SDS-PAGE under nonreducing conditions. N-Terminal sequences of the 65- and 48-kDa proteins were homologous to human lysosomal β-galactosidase and protective protein precursor, respectively. The purified sialidase complex also had carboxypeptidase activity. Both sialidase and carboxypeptidase activities were precipitated together by an antibody against chicken β-galactosidase. The complex reversibly dissociated into 120-kDa β-galactosidase dimer and 100-kDa carboxypeptidase dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. These findings indicate that chicken sialidase exists as a multienzyme complex, by which the sialidase activity appears to be stabilized.