Investigations for Mechanisms of Reactive Species Released from UPEs via Chemical Reactions

emerged

as

important

research

tools

with

high

peroxynitrite (ONOO−). It can also undergo spontaneous or

pharmacological potential. In our efforts to expand the

SOD-catalyzed dismutation with the formation of hydrogen

available toolbox of compounds that enable H2S-delivery,

peroxide (H2O2) and thus it is a precursor of other

polysulfides as well as caged thiocarbamate motifs. The

various pathologies. Due to its reactivity and short lifetime

we have recently demonstrated H2S release from synthetic thiocarbamate-based H2S donors function through the initial

biologically relevant oxidants playing an important role in in vivo, its detection and quantitation is difficult and

release of carbonyl sulfide (COS), which is quickly

demands special and sensitive techniques. One of the

converted to H2S by carbonic anhydrase (CA), which is

approaches is the use of fluorogenic probes, the compounds

expressed widely in mammalian systems. One added benefit

which themselves are not fluorescent but in the reaction

of such systems is that they can be tuned to release COS/H2S

with superoxide are oxidized to the fluorescent products

in response to specific biological or biorthogonal stimuli,

that can be directly detected. Among various profluorescent

thus providing a broad platform for tuning the donor motifs

probes available hydroethidine (HE) seems to be a gold

for specific applications. This presentation will focus on

standard for detection of superoxide in biological systems.

recent work from our lab aimed at (1) diversifying available

In the presence of superoxide radical anion, HE undergoes

COS/H2S donor core motifs and (2) preparing and applying

oxidative transformation into 2-hydroxyethidium (2-OH-

biologically-relevant

production. In the reaction with other radical, one-electron

new COS donors activated by specific stimuli, including small

molecules,

bio-orthogonal

triggers, and enzymatic reactions. In addition, we will also provide early insights into mitochondrial bioenergetics measurements that suggest that COS may exert different

E+),

a

specific

marker

of

superoxide

radical

anion

oxidants, HE is oxidatively transformed into ethidine and dimeric products. Here we present a spectroscopic and chemical characterization of new analogue of hydroethidine

activities than H2S in certain systems, thus highlighting the

- N,N,N,N-tetramethylhydroethidine (TMHE).

molecule.

DOI: 10.1016/j.freeradbiomed.2017.10.090

potential biological roles of this under-investigated small

DOI: 10.1016/j.freeradbiomed.2017.10.089

78 77

Investigations for Mechanisms of Reactive Species Released from UPEs via Chemical

N,N,N,N-tetramethylhydroethidine

Reactions

(TMHE) - in search for Novel Probes for the Detection of Superoxide Radical Anion Adam Sikora1, Radoslaw Michalski1, Adrianna Mesjasz1, Micael

Hardy2,

Olivier

Balaraman Kalyanaraman 1

Ouari2,

Jacek

Zielonka3,

and

3

Mika Tada1 1

Tohoku Institute of Technology, Japan

Conventional

oxidative

stress

markers,

including

malondialdehyde and 1-Nonanol are derived from the

Institute of Applied Radiation Chemistry, Lodz University

oxidation of unsaturated fatty acids. Lipid-derivers free radicals have been studied to cause damages to bio

of Technology, Poland 2

Aix Marseille Univ, CNRS, France, France

membranes, proteins and other biomolecules. In addition,

3

Medical College of Wisconsin, Milwaukee, USA

decompositions of lipid hydroperoxides are known to release excited triplet states of biomolecules composed with

The superoxide radical anion is an important reactive

carbonyl groups. On the other hand, ultra-weak photon

species produced by one-electron reduction of molecular

emissions (UPEs) are important to monitor in the redox

oxygen. The main sources of superoxide radical anion in vivo

status because UPEs have been used as biomarkers for non-

are the enzymes NADPH oxidases (NOX) and mitochondria

invasive

(mitochondrial electron transport chain). Superoxide radical

sometimes

anion reacts rapidly with nitric oxide with the formation of

composed with various wave lengths. In this study, we

64

SfRBM 2017

imaging confuse

techniques oxidative

[1-3]. stressors,

However,

UPEs

because

UPEs

investigated mechanisms of reactive species released from UPEs via chemical reactions of bio pigments (such as melanin) by qualitative analysis and polychromatic spectral pattern analysis. Acknowlegements: This work was supported by Grant-in-Aid of a private university (S1312001) from MEXT, Japan. I am deeply grateful to Prof. Masaki Kobayashi (Tohoku Institute of Technology, Japan), Dr. Yong-Ming Xie (Senior president

Sephs1 has a corresponding, but rather a shorter sequence Ala74-Ala114

(designated

as

int1).

The

in

vivo

complementation activity of Sephs2 was completely lost when the int2 sequence was substituted for the int1 sequence. Conversely, Sephs1 gained the SEPHS activity when the int1 region was replaced with int2 sequence in addition to the Thr to Cys substitution at the active site. Our results demonstrated for the first time that structural

his

requirement of mammalian Sephs includes the proline-rich

[1]E. van Wijk, M. Kobayashi, R. van Wijk, J. van der Greef,

the int2 sequence. Interestingly, co-expression of Sephs1

in

Asia,

PerkinElmer

Co.,

Ltd),

thank

you

for

collaborating and technical advices.

has not such an internal sequence region corresponding to

References: PLoS ONE 8, 12, e84579 (2013) [2]M. He, E. Van Wijk, R. Berger, et al., Mediators of Inflammation2015, 543541,1-11 (2015) [3]M. Kobayashi, T. Iwasa, M. Tada, J. Photochem. Photobiol. B: Biology 159, 186-190 (2016)

gene under varied concentrations of L-arabinose lead to gradual suppression of selD complementation by Sephs2Cys gene in E. coli. The hypothesis that the inactive homologue Sephs1 can suppress Sephs2Cys activity is also supported by in vitro pull-down assay and yeast-2-hybrid assay, which both indicated binding of Sephs1 to Seph2Cys.

DOI: 10.1016/j.freeradbiomed.2017.10.091

DOI: 10.1016/j.freeradbiomed.2017.10.092

79 An Internal Sequence Region Catalytically Essential for the Human Selenophosphate Synthetase 2 Muneaki Takahata1, Michiko Nemoto1, Kenji Inagaki1, and Takashi Tamura1 1

int2 region, although bacterial SEPHS including E. coli SelD

80 Lipofuscin Generated by UVA Exposure makes Human Skin Keratinocytes Sensitive to Visible Light Paulo Newton Tonolli1, Orlando Chiarelli Neto2, Carolina Santacruz-Perez1,

Okayama University, Japan

Watanabe , 1

Mammalian homologues, Sephs1 and Sephs2, differ greatly in their ability to complement selD mutation in E. coli.

Helena

Divinomar

Couto

Junqueira1,

Severino , 1

Felipe

Ii-Sei Gustavo

Ravagnani1, Waleska Kerllen Martins3, and Maurício S. Baptista1

Sephs2 is a selenoenyzme that has the Sec60 residue in the

1

University of São Paulo, Brazil

active site. Sephs2 retained the in vivo complementation

2

Centro Universitário do Espírito Santo, Brazil

when the Sec residue was replaced with Cys. Therefore,

3

Universidade de Santo Amaro, Brazil

Sephs2Cys gene allowed the production of the bacterial selenoenzyme, formate dehydrogenases, in a selD mutant of

The effects of UV radiation on skin cells are well known,

E. coli. In contrast, Sephs1 has a Thr residue at the

while the effect of other fractions of the solar spectrum

corresponding active site, and is an inactive catalysis

remains ignored. Recently, we observed that both neonatal

producing no complementation effect in E. coli selD mutant.

primary

Although Sephs2 and Sephs1 share a high homology in their

immortalized human keratinocytes (HaCaT) cells exposed to

amino acid sequences, Sephs1-Thr29Cys mutant did not

UVA and after exposure to visible light had reduction in cell

complement selD mutation in E. coli, suggesting that the

viability, increasing level of redox misbalance as monitored

lack of the active site Cys was not the sole reason for the

by the increase in DCF fluorescence, and the generation of

inert catalysis. Sephs2 gene encodes an internal Pro-rich

singlet oxygen. We also detected strand breaks of nuclear

sequence spanning Glu43-Arg83 (designated as int2), while

DNA and premutagenic Fpg and Endo III-sensitive DNA

SfRBM 2017

normal

human

keratinocyte

(NHK)

and

in

65