Myron K. Jacobson

Endogenous UVA-photosensitizers: mediators of skin photodamage and novel targets for skin photoprotection

Endogenous chromophores in human skin serve as photosensitizers involved in skin photocarcinogenesis and photoaging. Absorption of solar photons, particularly in the UVA region, induces the formation of photoexcited states of skin photosensitizers with subsequent generation of reactive oxygen species (ROS), organic free radicals and other toxic photoproducts that mediate skin photooxidative stress. The complexity of endogenous skin photosensitizers with regard to molecular structure, pathways of formation, mechanisms of action, and the diversity of relevant skin targets has hampered progress in this area of photobiology and most likely contributed to an underestimation of the importance of endogenous sensitizers in skin photodamage. Recently, UVA-fluorophores in extracellular matrix proteins formed posttranslationally as a consequence of enzymatic maturation or spontaneous chemical damage during chronological and actinic aging have been identified as an abundant source of light-driven ROS formation in skin upstream of photooxidative cellular stress. Importantly, sensitized skin cell photodamage by this bystander mechanism occurs after photoexcitation of sensitizers contained in skin structural proteins without direct cellular photon absorption thereby enhancing the potency and range of phototoxic UVA action in deeper layers of skin. The causative role of photoexcited states in skin photodamage suggests that direct molecular antagonism of photosensitization reactions using physical quenchers of photoexcited states offers a novel chemopreventive opportunity for skin photoprotection.

Poly(ADP-ribose) is required for spindle assembly and structure

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs). Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function. Here we show that PAR is enriched in the spindle and is required for spindle function—PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

Identification of α-dicarbonyl scavengers for cellular protection against carbonyl stress

Abstract Tissue deterioration and aging have long been associated with the accumulation of chemically induced protein and DNA damage. Reactive oxygen species (ROS) and reactive carbonyl species (RCS), especially α-dicarbonyl compounds, are key mediators of damage caused by oxidative stress, glycation, and UV-irradiation. The toxic effects of ROS are counteracted in vivo by antioxidants and antioxidant enzymes, and the deleterious effects of one RCS, methylglyoxal, are counteracted by a ubiquitous glyoxalase system. Carbonyl stress as a result of toxic effects of various mono-dicarbonyls (e.g. 4-hydroxynonenal) and α-dicarbonyls (e.g. glyoxal and deoxyosones) cannot be directly antagonized by antioxidants, and only a small number of biological carbonyl scavengers like glutathione (GSH) have been identified to date. We have developed a new screening method for the identification of carbonyl scavengers using a rapid glycation system that proceeds independent of oxygen and therefore, excludes identification of inhibitory compounds acting as antioxidants. Using this screening assay adapted to 96-well microtiter plates, we have identified the cysteine derivative 3,3-dimethyl- d -cysteine as a potent inhibitor of non-oxidative advanced glycation. Comparative kinetic analyses demonstrated the superior α-oxoaldehyde-scavenging activity of d -penicillamine over that of aminoguanidine. d -Penicillamine traps α-oxoaldehydes by forming a 2-acylthiazolidine derivative as shown by structure elucidation of reaction products between d -penicillamine and methylglyoxal or phenylglyoxal. We demonstrated that upon co-incubation, d -penicillamine protects human skin keratinocytes and fibroblasts (CF3 cells) against glyoxal- and methylglyoxal-induced carbonyl toxicity. Our research qualifies α-amino-β-mercapto-β,β-dimethyl-ethane as a promising pharmacophore for the development of related α-dicarbonyl scavengers as therapeutic agents to protect cells against carbonyl stress.

Human poly(ADP-ribose) glycohydrolase (PARG) gene and the common promoter sequence it shares with inner mitochondrial membrane translocase 23 (TIM23)

Poly(ADP-ribosyl)ation is a posttranslational protein modification mediated by members of the poly(ADP-ribose) polymerase (PARP) family. The ADP-ribose polymers, synthesized by the diverse PARP enzymes by cleavage of NAD(+), are involved in the regulation of multiple cellular functions. At present, only a single enzyme, poly (ADP-ribose) glycohydrolase (PARG), has been identified to catalyze ADP-ribose polymer hydrolysis in the cell causing a rapid turnover of the biopolymer which may ultimately result in lethal depletion of cellular NAD(+) pools. In this study, we describe the construction of the first human PARG cDNA clone by reverse transcription of CF3 human fibroblast RNA. Using the NCBI Genome BLAST program, the human PARG gene was mapped to chromosome 10 (10q11.23) in agreement to earlier results obtained by in situ hybridization. In vitro coupled transcription and translation of the cDNA yielded several specific bands in the range of 111-85 kDa, indicating possible usage of alternative translation initiation sites. The gene structure was characterized by further detailed computational analyses. The open reading frame consists of 18 exons and 17 introns with exons 9 to 14 forming the catalytic center of the enzyme and exons 1 to 3 encoding the putative regulatory domain. We show that the human PARG gene shares a 470-bp common promoter region with the inner mitochondrial membrane translocase 23 (TIM23). The human bidirectional promoter region was cloned and expression studies in transiently transfected HEK293 cells was performed using an EGFP-luciferase reporter fusion gene (GFL) to quantify transcription activation in both directions. The activity of the promoter was found to be 3.7 fold higher for TIM23 than for PARG, indicating that the two genes are expressed at different levels, although coregulation of the two genes remains an interesting possibility.

Optimizing the energy status of skin cells during solar radiation

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.

Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of alpha(1-->2) or alpha(1-->2) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG signature sequence [vDFA-X3-GGg-X6-8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610-795 in bovine PARG.

A topical lipophilic niacin derivative increases NAD, epidermal differentiation and barrier function in photodamaged skin

Abstract:u2002 The effects of myristyl nicotinate (MN), a nicotinic acid derivative designed to deliver nicotinic acid to skin without vasodilatation, on subjects with photodamaged skin have been studied. MN increased skin cell nicotinamide adenine dinucleotide (NAD) by 25% (Pu2003=u20030.001) demonstrating effective delivery of nicotinic acid to skin. Relative to placebo, MN treatment of photodamaged facial skin increased stratum corneum thickness by approximately 70% (Pu2003=u20030.0001) and increased epidermal thickness by approximately 20% (Pu2003=u20030.001). In two separate studies, MN treatment increased rates of epidermal renewal by 6% (Pu2003=u20030.003) to 11% (Pu2003=u20030.001) and increased the minimal erythemal dose by 8.9 (Pu2003=u20030.07) and 10% (Pu2003=u20030.05) relative to placebo. MN treatment resulted in reductions in the rates of transepidermal water loss (TEWL) of approximately 20% relative to placebo on cheeks (Pu2003=u20030.012) and arms (Pu2003=u20030.017) of study subjects. Results of a tape stripping challenge before and after MN treatment demonstrated a significant correlation (Pu2003=u20030.03) between increased skin NAD content and resistance to changes in TEWL for MN treated but not placebo subjects. Rates of TEWL changed more rapidly and to a greater extent in atopic subjects compared with normal subjects. The results indicate that MN enhances epidermal differentiation and barrier function in skin, suggesting that this method of nicotinic acid delivery may prove useful in limiting progression of actinic skin damage and possibly in treating other conditions involving skin barrier impairment.

A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence.

Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.

Identification of Quenchers of Photoexcited States as Novel Agents for Skin Photoprotection

Photooxidative stress is a key mechanism in UVA-induced skin photodamage. Photoexcited states of endogenous UVA chromophores such as porphyrins, melanin precursors, and cross-link-fluorophores of skin collagen exert skin photodamage by direct reaction with substrate molecules (type I photosensitization) or molecular oxygen (type II), leading to formation of reactive oxygen species. Based on our previous research on the role of photoexcited states of endogenous skin chromophores as sensitizers of photooxidative stress, we describe here the identification of a novel class of chemopreventive agents for topical skin photoprotection: quenchers of photoexcited states (QPES). QPES compounds antagonize the harmful excited state chemistry of endogenous sensitizers by physical quenching, facilitating the harmless return of the sensitizer excited state to the electronic ground state by energy dissipation. To identify QPES compounds suitable for development, we designed a primary screening assay based on QPES suppression of photosensitized plasmid cleavage using conditions that exclude antioxidants. This screen is followed with a screen to test for nonsacrificial quenching of dye-sensitized singlet oxygen (1O2) formation by electron paramagnetic resonance detection of 2,2,6,6-tetramethyl-piperidine-1-oxyl, a stable free radical indicative of 1O2 formation. These initial screens identified a pyrrolidine pharmacophore with pronounced QPES activity, and l-proline and other noncytotoxic proline derivatives containing this pharmacophore were then screened for efficacy in cellular models of sensitized photodamage. These compounds showed QPES protection against dye-sensitized and psoralen-UVA-induced apoptosis and suppression of proliferation in cultured human skin keratinocytes and fibroblasts. Furthermore, QPES photoprotection of reconstructed full thickness human skin exposed to solar simulated light has been demonstrated.