Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy.

DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH(-) (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD(.-) and FADH(.) formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD(.-) (formed by ET from the tryptophan chain) in plant CRYs in about 1.5 µs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 µs into a state with spectral features that are different from both FADH(.) and FAD(.-) . Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

Prohepcidin binds to the HAMP promoter and autoregulates its own expression.

Hepcidin is the major regulatory peptide hormone of iron metabolism, encoded by the HAMP (hepcidin antimicrobial peptide) gene. Hepcidin is expressed mainly in hepatocytes, but is also found in the blood in both a mature and prohormone form. Although, the function of mature hepcidin and the regulation of the HAMP gene have been extensively studied, the intracellular localization and the fate of prohepcidin remains controversial. In the present study, we propose a novel role for prohepcidin in the regulation of its own transcription. Using indirect immunofluorescence and mCherry tagging, a portion of prohepcidin was detected in the nucleus of hepatocytes. Prohepcidin was found to specifically bind to the STAT3 (signal transducer and activator of transcription 3) site in the promoter of HAMP. Overexpression of prohepcidin in WRL68 cells decreased HAMP promoter activity, whereas decreasing the amount of prohepcidin caused increased promoter activity measured by a luciferase reporter-gene assay. Moreover, overexpression of the known prohepcidin-binding partner, α-1 antitrypsin caused increased HAMP promoter activity, suggesting that only the non-α-1 antitrypsin-bound prohepcidin affects the expression of its own gene. The results of the present study indicate that prohepcidin can bind to and transcriptionally regulate the expression of HAMP, suggesting a novel autoregulatory pathway of hepcidin gene expression in hepatocytes.

Conformational dynamics of titin PEVK explored with FRET spectroscopy.

The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.

Dynamic strength of titin's Z-disk end.

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity.

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.

Cardiac leiomodin2 binds to the sides of actin filaments and regulates the ATPase activity of myosin.

Leiomodin proteins are vertebrate homologues of tropomodulin, having a role in the assembly and maintenance of muscle thin filaments. Leiomodin2 contains an N-terminal tropomodulin homolog fragment including tropomyosin-, and actin-binding sites, and a C-terminal Wiskott-Aldrich syndrome homology 2 actin-binding domain. The cardiac leiomodin2 isoform associates to the pointed end of actin filaments, where it supports the lengthening of thin filaments and competes with tropomodulin. It was recently found that cardiac leiomodin2 can localise also along the length of sarcomeric actin filaments. While the activities of leiomodin2 related to pointed end binding are relatively well described, the potential side binding activity and its functional consequences are less well understood. To better understand the biological functions of leiomodin2, in the present work we analysed the structural features and the activities of Rattus norvegicus cardiac leiomodin2 in actin dynamics by spectroscopic and high-speed sedimentation approaches. By monitoring the fluorescence parameters of leiomodin2 tryptophan residues we found that it possesses flexible, intrinsically disordered regions. Leiomodin2 accelerates the polymerisation of actin in an ionic strength dependent manner, which relies on its N-terminal regions. Importantly, we demonstrate that leiomodin2 binds to the sides of actin filaments and induces structural alterations in actin filaments. Upon its interaction with the filaments leiomodin2 decreases the actin-activated Mg2+-ATPase activity of skeletal muscle myosin. These observations suggest that through its binding to side of actin filaments and its effect on myosin activity leiomodin2 has more functions in muscle cells than it was indicated in previous studies.

Flow cytometric analysis of DMBA-induced early in vivo ras expression.

According to recent publications 7,12-dimethylbenz[a]anthracene (DMBA) induces not only mammary cancer but also leukemia in Long-Evans (LE) rats. After treatment with DMBA, trisomy of the chromosome bearing N-ras and mutations in the codon 61 of different ras family genes are frequent. These alterations are already visible within 48 hours. Since there are very few data on ras genes' expression in the early stages of leukemogenesis, in our investigations LE rats were treated with DMBA and the expression of ras genes was measured within two days. DMBA was administered to outbred Long-Evans rats and the fluorescence intensity of the antibody recognizing the ras gene family was measured in femoral bone marrow cells 24 and 48 hours after the treatment. One of the bone marrow cell populations, separated by FSC and SSC, showed elevated ras gene expression at both 24 and 48 hours after the administration of the carcinogen. These results suggest that, besides the specific chromosomal aberrations and gene mutations, elevated ras gene expression could also be the marker of DMBA exposure.

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor.

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules.

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.

Reversible mechanical unzipping of amyloid beta-fibrils.

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid beta (Abeta) peptides 1-40 or 25-35, beta-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Abeta1-40 and Abeta25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the beta-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of beta-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.

Stretching and visualizing titin molecules: combining structure, dynamics and mechanics.

The details of the global and local structure and function of titin, a giant filamentous intrasarcomeric protein are largely undiscovered. Here we discuss a combination of bulk-solution and novel single-molecule techniques that may lend unique insights into titin's molecular dynamic, structural and mechanical characteristics.

Differential actin binding along the PEVK domain of skeletal muscle titin.

Parts of the PEVK (Pro-Glu-Val-Lys) domain of the skeletal muscle isoform of the giant intrasarcomeric protein titin have been shown to bind F-actin. However, the mechanisms and physiological function of this are poorly understood. To test for actin binding along PEVK, we expressed contiguous N-terminal (PEVKI), middle (PEVKII), and C-terminal (PEVKIII) PEVK segments of the human soleus muscle isoform. We found a differential actin binding along PEVK in solid-state binding, cross-linking and in vitro motility assays. The order of apparent affinity is PEVKII>PEVKI>PEVKIII. To explore which sequence motifs convey the actin-binding property, we cloned and expressed PEVK fragments with different motif structure: PPAK, polyE-rich and pure polyE fragments. The polyE-containing fragments had a stronger apparent actin binding, suggesting that a local preponderance of polyE motifs conveys an enhanced local actin-binding property to PEVK. The actin binding of PEVK may serve as a viscous bumper mechanism that limits the velocity of unloaded muscle shortening towards short sarcomere lengths. Variations in the motif structure of PEVK might be a method of regulating the magnitude of the viscous drag.