Berberine–DNA complexation: New insights into the cooperative binding and energetic aspects

The equilibrium binding of the cytotoxic plant alkaloid berberine to various DNAs and energetics of the interaction have been studied. At low ratios of bound alkaloid to base pair, the binding exhibited cooperativity to natural DNAs having almost equal proportions of AT and GC sequences. In contrast, the binding was non-cooperative to DNAs with predominantly high AT or GC sequences. Among the synthetic DNAs, cooperative binding was observed with poly(dA).poly(dT) and poly(dG).poly(dC) while non-cooperative binding was seen with poly(dA–dT).poly(dA–dT) and poly(dG–dC).poly(dG–dC). Both cooperative and non-cooperative bindings were remarkably dependent on the salt concentration of the media. Linear plots of ln K_a versus [Na⁺] for poly(dA).poly(dT) and poly(dA–dT).poly(dA–dT) showed the release of 0.56 and 0.75 sodium ions respectively per bound alkaloid. Isothermal titration calorimetry results revealed the binding to be exothermic and favoured by both enthalpy and entropy changes in all DNAs except the two AT polymers and AT rich DNA, where the same was predominantly entropy driven. Heat capacity values (ΔCp^o) of berberine binding to poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), Clostridium perfringens and calf thymus DNA were − 98, − 140, − 120 and − 110 cal/mol K respectively. This study presents new insights into the binding dependent base pair heterogeneity in DNA conformation and the first complete thermodynamic profile of berberine binding to DNAs.     

Structural insights into the cooperative binding of SeqA to a tandem GATC repeat

SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqAΔ(41–59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA–DNA complex also unveils additional protein–protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.     

Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects

Few information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant‐derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2‐phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations.     

In silico analysis of the cyanobacterial lectin scytovirin: new insights into binding properties

Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1–48 (SD1) and the second 49–95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1?+?SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.     

X-ray structure of human proMMP-1: new insights into procollagenase activation and collagen binding

Vertebrate collagenases, members of the matrix metalloproteinase (MMP) family, initiate interstitial fibrillar collagen breakdown. It is essential in many biological processes, and unbalanced collagenolysis is associated with diseases such as arthritis, cancer, atherosclerosis, aneurysm, and fibrosis. These metalloproteinases are secreted from the cell as inactive precursors, procollagenases (proMMPs). To gain insights into the structural basis of their activation mechanisms and collagen binding, we have crystallized recombinant human proMMP-1 and determined its structure to 2.2 A resolution. The catalytic metalloproteinase domain and the C-terminal hemopexin (Hpx) domain show the classical MMP-fold, but the structure has revealed new features in surface loops and domain interaction. The prodomain is formed by a three-helix bundle and gives insight into the stepwise activation mechanism of proMMP-1. The prodomain interacts with the Hpx domain, which affects the position of the Hpx domain relative to the catalytic domain. This interaction results in a "closed" configuration of proMMP-1 in contrast to the "open" configuration observed previously for the structure of active MMP-1. This is the first evidence of mobility of the Hpx domain in relation to the catalytic domain, providing an important clue toward the understanding of the collagenase-collagen interaction and subsequent collagenolysis.     

From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors.

Transmembrane signaling systems relay information from the exterior to the interior of a cell, through a series of complex protein-protein interactions and second messenger cascades. One such system consists of the G-protein-coupled receptors, which interact with G proteins upon ligand binding, and in turn activate an effector molecule. The receptor is the first component in this signaling cascade and is subject to considerable regulation. Recent studies have shown that these regulatory events can occur at the levels of receptor protein modification and receptor gene expression. Interestingly, some of these processes appear to be mediated by the same second messenger systems that these receptors activate, which leads to various forms of positive and negative feedback regulation.     

Molecular aspects of atherogenesis: new insights and unsolved questions

Summary The development of atherosclerotic disease results from the interaction between environment and genetic make up. A key factor in atherogenesis is the oxidative modification of lipids, which is involved in the recruitment of mononuclear leukocytes to the arterial intima – a process regulated by several groups of adhesion molecules and cytokines. Activated leukocytes, as well as endothelial mitochondria, can produce reactive oxygen species (ROS) that are associated with endothelial dysfunction, a cause of reduced nitric oxide (NO) bioactivity and further ROS production. Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are nuclear receptors significantly involved in the control of lipid metabolism, inflammation and insulin sensitivity. Also, an emerging role has been suggested for G protein coupled receptors and for the small Ras and Rho GTPases in the regulation of the expression of endothelial NO synthase (eNOS) and of tissue factor, which are involved in thrombus formation and modulation of vascular tone. Further, the interactions among eNOS, cholesterol, oxidated LDL and caveola membranes are probably involved in some molecular changes observed in vascular diseases. Despite the relevance of oxidative processes in atherogenesis, anti-oxidants have failed to significantly improve atherosclerosis (ATS) prevention, while statins have proved to be the most successful drugs.     

Phosphoproteomics: new insights into cellular signaling

Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far.     

The equine endometrosis: new insights into the pathogenesis

This paper describes the histomorphological and immunohistochemical characterisation of phenotypic variations of endometrosis as well as potential etiological factors which may influence disease progression. In total, 779 endometrial biopsies were examined. These biopsies were taken in the breeding and non-breeding season (n=509), on defined days during the estrous cycle (n=70) and before and after experimentally induced bacterial endometritis (n=200). In addition to conventional histopathology, selected biopsies were investigated using alcianblue staining as well as immunohistochemical methods for the detection of steroid hormone receptors, Ki-67-antigen, vimentin, desmin, fibronectin, smooth-muscle-alpha-actin and laminin. The equine endometrosis can be divided into a destructive and a non-destructive form. Based on the morphology of the stromal cells involved, an active or inactive state can be distinguished in fibrotic foci. In all types of endometrosis, fibrotic stromal cells show a distinctly reduced expression of steroid hormone receptors in comparison to the intact stroma, indicating their dedifferentiation. However, the steroid hormone receptor expression of involved glandular epithelia seems to depend on the activity of the fibrosis. These results suggest an independency of all fibrotic foci from the hormonal control mechanism of the uterus. The characteristical features of destructive endometrosis are a large number of smooth-muscle-alpha-actin containing myofibroblasts, a pronounced epithelial vimentin expression, excessive extracellular matrix accumulation and a progressive alteration of the basal lamina. Furthermore, the frequently seen cystic glandular dilatation and mechanical destruction of the uterine glands may occur due to the contractibility of the myofibroblasts involved. As shown in this study, a simultaneous endometritis can cause a temporary activation of fibrotic stromal cells. However, cyclic and seasonal endocrine changes seem to have no effects on progression of the disease. It can be concluded that the various types of endometrosis represent different stages in the fibrotic process, possibly leading to the destruction of the glands and subsequently resulting in the development of a stromal fibrosis.     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

Subnuclear organelles: new insights into form and function

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.     

Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.     

Capping protein: new insights into mechanism and regulation

Temporal and spatial control of the actin cytoskeleton are crucial for a range of eukaryotic cellular processes. Capping protein (CP), a ubiquitous highly conserved heterodimer, tightly caps the barbed (fast-growing) end of the actin filament and is an important component in the assembly of various actin structures, including the dynamic branched filament network at the leading edge of motile cells. New research into the molecular mechanism of how CP interacts with the actin filament in vitro and the function of CP in vivo, including discoveries of novel interactions of CP with other proteins, has greatly enhanced our understanding of the role of CP in regulating the actin cytoskeleton.     

Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism

Adenosylcobalamin-dependent isomerases catalyze a variety of chemically difficult 1,2-rearrangements that proceed through a mechanism involving free radical intermediates. These radicals are initially generated by homolysis of the cobalt-carbon bond of the coenzyme. Recently, the crystal structures of several of these enzymes have been solved, revealing two modes of coenzyme binding and highlighting the role of the protein in controlling the rearrangement of reactive substrate radical intermediates. Complementary data from kinetic, spectroscopic and theoretical studies have produced insights into the mechanism by which substrate radicals are generated at the active site, and the pathways by which they rearrange.     

Mitochondria and dopamine: new insights into recessive parkinsonism

Recessively inherited mutations in parkin, DJ-1, and PINK1 have recently been linked to familial forms of parkinsonism. These syndromes are often clinically indistinguishable from Parkinson's disease, as similar neuronal groups, notably dopaminergic neurons, are selectively affected. Studies of the functions of these gene products may provide insights into the pathogenic mechanisms underlying the selective degeneration of dopaminergic neurons. Emerging evidence that one or several of these genes play important roles in mitochondrial function and the dopaminergic system suggests that these events may be early steps of the pathophysiological changes of the disease. This review will summarize recent advances in our understanding of these gene products, with emphasis on the surprising convergence of their functions.     

Neonatal diabetes: new insights into aetiology and implications

Neonatal diabetes mellitus (NDM) is defined as hyperglycaemia occurring in the first few weeks of life. It can be either transient (TNDM) or permanent (PNDM), and until recently, little was known about the condition. A cohort of 30 infants with a history of TNDM has been studied, and findings have suggested that NDM does not have the same aetiology as classical type 1 childhood diabetes. Uniparental isodisomy of chromosome 6 and an unbalanced duplication of paternal chromosome 6 have both been described as a genetic basis for TNDM in over 75% of the cases. In addition, cerebellar hypoplasia and Walcott-Rallison syndrome have been associated with PNDM, suggesting an autosomal recessive inheritance pattern; furthermore, a mutation in the gene insulin promoter factor 1 has been identified as a cause of pancreatic agenesis in PNDM. In the long term, TNDM may reduce beta cell functional capacity and present a predisposition to type 2 diabetes mellitus.     

Nuocytes and beyond: new insights into helminth expulsion

T helper 2 (Th2) responses, characterized by the expression of the type-2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, are essential for the control of parasitic helminth infections and also drive the pathogenesis of allergy and asthma. Such responses are initiated, maintained and regulated, in part, by an array of innate effector cells and cytokines. However, relatively little is known about how the initiation of type-2 immune responses occurs in vivo. The recent discovery, using helminth models, of several novel innate immune cells capable of shaping type-2 immune responses allows us to reflect on the progress made in this area. It also affords us the opportunity to highlight the diversity of immune responses that can be driven by innate cells responding rapidly to early cytokine cues.