UVA-induced upregulation of progerin suppresses 53BP1­mediated NHEJ DSB repair in human keratinocytes via progerin-lamin A complex formation.

Ultraviolet (UV) radiation is the primary risk factor underlying photoaging and photocarcinogenesis. Mounting research has focused on the role of DNA damage response pathways in UV-induced double-strand break (DSB) repair. In the present study, we hypothesized that UVA-induced aberrant progerin upregulation may adversely affect p53-binding protein 1 (53BP1)-mediated non-homologous end joining (NHE) DSB repair in human keratinocytes. Basal cell carcinoma (BCC) tumors and matching normal skin tissue were sampled (n=200) to investigate whether human keratinocytes display dysregulated progerin expression as a function of advancing age and BCC status. Newborn foreskin samples (n=9) were used as a source for primary keratinocyte cultures. We investigated the effects of UVA radiation on progerin and lamin A expression as well as the effects of the silencing of progerin on lamin A protein expression in UVA-irradiated keratinocytes. We investigated whether blocking progerin­lamin A interaction was able to rescue UVA-induced lamin A protein downregulation, 53BP1 downregulation and 53BP1-mediated NHEJ DSB repair activity. Progerin upregulation in adult keratinocytes was associated with advancing age, not BCC status. In vitro, UVA exposure significantly upregulated progerin expression by favoring alternative LMNA gene transcript splicing. UVA exposure significantly downregulated free (unbound) lamin A protein levels via progerin-lamin A complex formation. UVA exposure significantly decreased 53BP1 protein levels via enhanced progerin-lamin A complex formation. UVA-induced progerin­lamin A complex formation was largely responsible for suppressing 53BP1-mediated NHEJ DSB repair activity. The present study is the first to demonstrate that UVA-induced progerin upregulation adversely affects 53BP1-mediated NHEJ DSB repair in human keratinocytes via progerin­lamin A complex formation.

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

Effects of gamma irradiation on collagen damage and remodeling.

PURPOSE: To evaluate the dose-time dependences of structural changes occurring in collagen within 24 hours to three months after gamma-irradiation at doses from 2-40 Gy in vivo. MATERIALS AND METHODS: Rat's tail tendon was chosen as in vivo model, with its highly ordered collagen structure allowing the changes to be interpreted unambiguously. Macromolecular level (I) was investigated by differential scanning calorimetry (DSC); fibers and bundles level (II) by laser scanning microscopy (LSM), and bulk tissue microstructural level (III) by cross-polarization optical coherence tomography (CP-OCT). RESULTS: For (I), the formation of molecular cross-links and breaks appeared to be a principal mechanism of collagen remodeling, with the cross-links number dependent on radiation dose. Changes on level (II) involved primary, secondary and tertiary bundles splitting in a day and a week after irradiation. Bulk collagen microstructure (III) demonstrated early widening of the interference fringes on CP-OCT images observed to occur in the tendon as result of this splitting. At all three levels, the observed collagen changes demonstrated complete remodeling within ∼ a month following irradiation. CONCLUSION: The time course and dose dependencies of the observed collagen changes at different levels of its hierarchy further contribute to elucidating the role of connective tissue in the radiotherapy process.

Photo-induced reversible structural transition of cationic diphenylalanine peptide self-assembly.

The photo-induced self-assembly of a cationic diphenylalanine peptide (CDP) is investigated using a photoswitchable sulfonic azobenzene as the manipulating unit. A reversible structural transition between a branched structure and a vesicle-like structure is observed by alternating between UV and visible light irradiation.

Mini-beam collimator enables microcrystallography experiments on standard beamlines.

The high-brilliance X-ray beams from undulator sources at third-generation synchrotron facilities are excellent tools for solving crystal structures of important and challenging biological macromolecules and complexes. However, many of the most important structural targets yield crystals that are too small or too inhomogeneous for a ;standard' beam from an undulator source, approximately 25-50 microm (FWHM) in the vertical and 50-100 microm in the horizontal direction. Although many synchrotron facilities have microfocus beamlines for other applications, this capability for macromolecular crystallography was pioneered at ID-13 of the ESRF. The National Institute of General Medical Sciences and National Cancer Institute Collaborative Access Team (GM/CA-CAT) dual canted undulator beamlines at the APS deliver high-intensity focused beams with a minimum focal size of 20 microm x 65 microm at the sample position. To meet growing user demand for beams to study samples of 10 microm or less, a ;mini-beam' apparatus was developed that conditions the focused beam to either 5 microm or 10 microm (FWHM) diameter with high intensity. The mini-beam has a symmetric Gaussian shape in both the horizontal and vertical directions, and reduces the vertical divergence of the focused beam by 25%. Significant reduction in background was achieved by implementation of both forward- and back-scatter guards. A unique triple-collimator apparatus, which has been in routine use on both undulator beamlines since February 2008, allows users to rapidly interchange the focused beam and conditioned mini-beams of two sizes with a single mouse click. The device and the beam are stable over many hours of routine operation. The rapid-exchange capability has greatly facilitated sample screening and resulted in several structures that could not have been obtained with the larger focused beam.

Radiation damage in macromolecular cryocrystallography.

X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.

Radiation driven collapse of protein crystals.

During coherent X-ray diffraction measurements on crystals of ferritin at room temperature using monochromatic undulator radiation from the Advanced Photon Source, a sudden lattice contraction was observed following a characteristic latent period and ultimately leading to the collapse of the crystal. The progression of this collapse is analysed using a two-state Hendricks-Teller model. It reveals that 55% of the layers collapse by 1.6% before the crystal completely stops diffracting.

Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants.

In small-angle X-ray scattering experiments at high-brilliant synchrotron sources, protein aggregation results from radiation damage. The radiation-induced aggregation of lysozyme in solution was qualitatively evaluated based on forward scattering and radii of gyration. The scattering did not change below 400 Gy and increased exponentially above this dose. The aggregation is only seen beyond the critical dose rate, and the 'dilution effect' known in radiology was also observed. Mass spectroscopy of the lysozyme solution exposed to a monochromatic X-ray beam did not show any cleavage of the polypeptide chain. Small-angle X-ray scattering patterns suggested that the radiation-induced aggregation should be a non-specific association of intact lysozyme, without substantial alterations of the folding topologies. It was found that the addition of small amounts of cryoprotectants, such as glycerol, ethylene glycol and sucrose, effectively reduced the radiation damage. Glycerol and ethylene glycol were identically effective in the 100 mM concentration range. A similar effective concentration was observed for sucrose. The damage reduction by the cryoprotectants was mainly ascribed to changes in the protein-protein interactions, and rarely to decreases in the diffusion rates of activated species.