Tài liệu Báo cáo khoa học: Oral Presentations Integration of Metabolism and Survival pdf

Oral Presentations

Integration of Metabolism and Survival

OP-1

The ankyrin repeat and SOCS box-containing

protein Asb-9 targets creatine kinase B for

degradation

M. A. Debrincat1

, J. G. Zhang2

, T. A. Willson3

, B. T. Kile3

,

S. L. Masters2

, L. M. Connolly4

, R. J. Simpson4

, H. M. Martin2

,

N. A. Nicola2 and D. J. Hilton3

1

Cancer and Haematology/Molecular Medicine, The Walter

and Eliza Hall Institute of Medical Research, Melbourne,

Australia,2

Cancer and Haematology, The Walter and Eliza Hall

Institute of Medical Research, Melbourne, Australia,3

Molecular

Medicine, The Walter and Eliza Hall Institute of Medical

Research, Melbourne, Australia,4

The Joint Proteomics Laboratory

of the Walter and Eliza Hall Institute of Medical Research and

Ludwig Institute for Cancer Research, Melbourne, Australia.

E-mail: [email protected]

The suppressors of cytokine signalling (SOCS) proteins inhibit

cytokine signalling by direct interaction with Janus kinases

(JAKs) or activated cytokine receptors. In addition to the N-ter￾minal and SH2 domains that mediate these interactions, SOCS

proteins contain a C-terminal SOCS box. Evidence suggests

SOCS box-containing proteins act as part of an elongin-cullin￾SOCS (ECS) E3 ubiquitin ligase complex, marking target pro￾teins for degradation. The specificity of the complex is deter￾mined by the protein interaction motif located upstream from the

SOCS box. A number of other protein families that possess a

SOCS box have been identified, the largest of which are the ank￾yrin repeat and SOCS box-containing Asbs. While it is known

that the SOCS proteins are involved in the negative regulation of

cytokine signalling, the biological and biochemical functions of

the Asbs are undefined. To understand the functional role of Asb

proteins, a proteomic approach was implemented and creatine

kinase B (CKB) was identified as a specific binding partner of

Asb-9. Transfection of increasing concentrations of a tagged

Asb-9 construct into 293T cells increased the polyubiquitination

of CKB and resulted in a concomitant decrease in total CKB

levels within the cell. The targeting of CKB for degradation by

Asb-9 was entirely SOCS box-dependent. The interaction has

been confirmed in vivo and suggests that Asb-9 may act as a

specific ubiquitin ligase-regulating CKB abundance.

OP-2

Signal transduction of cytokinine

M. Gilmanov1

, S. Ibragimova1

, Sv. Sadykova1

, Zh. Basygaraev1

and A. Sabitov2

1

Laboratory of Enzyme Structure and Regulation, Aithozhin’s

Institute of Molecular Biology and Biochemistry,2

Department of

Chemistry, Al-Faraby’s Kazakn National University.

E-mail: [email protected]

The cytokinine the most important phytohormone, which is con￾trolling the division of the plant, cells. We carried out the investi￾gation of cytokinine signal transduction. We were discovered the

most important participant of cytokinine signal transduction.

This participant was purified from germinated wheat grains by

hydrophobic chromathography on octyl-sepharose 4B CL and by

reversed-phase chromatography on RP-18 type column. Thus we

were named the isolated substance as secondary cytokinine hor￾mone or shorter mediator of cytokinine (MC). The MC is very

powerful phytohormone as it shows the physiological activity at

concentration 1000 times less than cytokinine. In contrast of

cytokinine the MC appears own biochemical activities. For exam￾ple, MC is activated NADP-glutamate dehydrogenase and

H+ATP-ase, while the cytokinine not be able to activate this

enzymes. The purified MC is competitive repressed the binding

of tritium-labeled fusicoccine with fusicoccine receptors on plas￾matic membrane winch were isolated from roots of Zea mays

sprouts. It was shown that the binding of MC with fusicoccine

receptors led to increasing the level of cytoplasmic calcium ions.

Then calcium ions by participation of inositides is activated the

proteinkinase C. Proteinkinase C was isolated by gel chromatog￾raphy on sephacryle S-300 column and by ion-exchange chroma￾tography on DE-52 column. This enzyme is the last participant

of the signal transduction of cytokinine.

OP-3

Skeletal muscle elongation factor 2

phosphorylation during contraction:

mechanism of regulation

A. J. Rose, J. B. Kobberø, T. J. Alsted, T. E. Jensen and

E. A. Richter

Copenhagen Muscle Research Centre, Department of Human

Physiology, Institute of Exercise & Sport Sciences, Copenhagen,

Denmark. E-mail: [email protected]

Very little is known about the effect of exercise on the molecular

regulation of polypeptide synthesis in skeletal muscle. Here, the

effect of contractions on skeletal muscle eukaryotic elongation

factor 2 (eEF2) phosphorylation and eEF2 kinase activity was

investigated. In response to contractions in situ, there was a rapid

(i.e. 15s) fivefold increase in eEF2 phosphorylation at Thr56 in

the contracting gastrocnemius muscle of rats that was maintained

at this level during 30 min of contractions, with no change in the

non-stimulated contralateral muscle. Furthermore, eEF2 phos￾phorylation was higher in both soleus and extensor digitorum

longus muscles of mice when contracted ex vivo, indicating that

the mechanism behind this increase is related to local factors. No

change in in vitro eEF2 kinase activity was observed in the con￾tracted rat muscles at any time-point or when measured at pH

6.8 versus 7.2. Furthermore, the increase in eEF2 phosphoryla￾tion occurred at a time before any change in AMPK activity was

observed and was normal in contracting muscles of mice expres￾sing non-functional AMPK. However, Ca2+-calmodulin potently

increased the activity of skeletal muscle eEF2 kinase when meas￾ured in vitro. Taken together, these data indicate that the inhibi￾tion of protein synthesis in contracting muscle may arise from

phosphorylation of eEF2 via a Ca2+-calmodulin-eEF2 kinase

cascade.

Abstracts

42

Integration of Defence and Survival

OP-4

Investigation of multidrug resistance in

docetaxel and doxorubicin-resistant MCF-7 cell

lines

O¨. Darcansoy _

Is¸ eri1

, M. Demirel Kars1

, U. Gu¨ndu¨z

1 and

F. Arpacı

2

1

Department of Biological Sciences, Middle East Technical

University, Ankara, Turkey,2

Department of Oncology, Gu¨lhane

Military Academy School of Medicine, Ankara, Turkey.

E-mail: [email protected]

Ineffectiveness of anticancer drugs during chemotherapy or recur￾rence of malignancy after therapy is a frequently observed situation

in cancer chemotherapy. Multidrug resistance (MDR) phenomenon

is defined as the resistance of tumor cells to various cytotoxic drugs.

It is a major impediment to successful treatment of breast cancer

using chemotherapy. Cancer cells either strengthen the already pre￾sent systems necessary for the removal of toxins from cells or acquire

resistance to cytotoxic drugs. Members of the ATP-binding cassette

(ABC) transporter superfamily have an important role in MDR.

Among these, proteins coded by the ABCB1 (MDR1), ABCC1

(MRP1), and ABCG2 (BCRP) genes are the most important trans￾porters related to MDR phenotype. In this study, effects of expres￾sion levels of the MDR1, MRP1, BCRP genes on the development

of docetaxel and doxorubicin resistance using a model MCF-7

breast carcinoma cell line is evaluated. Docetaxel and doxorubicin

were applied to cell culture in dose increaments and resistant sub￾lines were developed. Cytotoxicity analysis of drugs was performed

in wild type and developed resistant sublines to test development

of resistance. Total RNA was isolated from cells, converted to

cDNA and amplified by using gene (MDR1, MRP1, and BCRP)-

specific primers by RT-PCR. Western blot analysis and immuno￾staining were performed to determine the related protein levels.

OP-5

GSK3: identification of a novel mechanism

controlling inflammation in the brain

E. Beurel1

, S. Michalek2 and R. Jope1

1

Department of Psychiatry and Behavioral Neurobiology,

University of Alabama at Birmingham, Birmingham, AL,

USA,2

Department of Microbiology, University of Alabama at

Birmingham, Birmingham, AL, USA. E-mail: [email protected]

Controlling inflammation is a major challenge in the brain where

inflammation has devastating consequences because, since dam￾aged neurons cannot be replaced, neuroinflammation contributes

to neurodegenerative diseases. In response to lipopolysaccharide

(LPS), brain microglia, and astrocytes activate cytokines produc￾tion, such as IL-6. Glycogen synthase kinase-3 (GSK3) regulating

transcription factors, was studied as a regulator of neuroinflam￾mation. In mouse primary astrocytes, we examined if GSK3-

regulated IL6 production stimulated by LPS and its amplified

production caused by co-administered interferon-c. Both LPS￾induced IL-6 production and also its potentiation by interferon-c

are highly dependent on GSK3. With both IL-6 production was

abolished by GSK3 inhibitors, demonstrating the key role of

GSK3 in neuroinflammation. The mechanism of LPS-induced

IL-6 production potentiated by interferon-c was due to GSK3

activation and nuclear depletion of GSK3. This suggests that

interferon-c plus LPS potentiates IL-6 production by activating

cytosolic GSK3 which can control the activation of transcription

factors that activate IL-6 expression. Chromatin immunoprecipi￾tation is being used to identify the transcriptional targets of

GSK3, such as NF-jB that regulate IL-6 production. This study

identified GSK3 as a key regulator of neuroinflammation, estab￾lishing that GSK3 inhibitors provide a new strategy to counteract

the devastating effects of neuroinflammation.

Rhythmic Signals: the Setting of Biological Time

OP-6

Computational search of the interaction

between melanopsin and cryptochrome-2

proteins

E. B. Unal1

, B. Erman2 and I. H. Kavakli2

1

Computational Sciences and Engineering, Koc University, Istan￾bul, Turkey,2

Chemical and Biological Engineering, Koc University,

Istanbul, Turkey. E-mail: [email protected]

Circadian rhythms are the biological processes that oscillate in the

biochemical, physiological and behavioral functions of organisms

with a periodicity of approximately 24 h without any external cues.

In mammals, circadian rhythm is generated by molecular clock,

which is located at suprachiasmatic nuclei (SCN) part of brain.

Circadian rhythm is reset by external factors such as light. The

cryptochromes,which was first discovered in Arabidopsis,are the

blue-light photoreceptors. They absorb light and transmit the elec￾tromagnetic signal to blue-light dependent of signal transduction.

In mammals, the cryptochromes and melanopsin have been pro￾posed as circadian photoreceptor pigments that exist in the inner

retina to transmit signal to the SCN to tell the time of day. Both

humans and mice have two cryptochrome proteins; CRY1 and

CRY2. CRY2 is mostly expressed in retinal cells. Based on current

evidence we propose that CRY2 may interact with melanopsin to

mediate the light dependent signal-transduction in mammals.We

have taken both computational and experimental approaches in

order to show possible interaction between them during the circa￾dian photoreception. First, we have predicted 3-D structure of

cryptochrome using EsyPred, Robetta programs.Then, we have

shown that both cryptochrome and melanopsin may interact in

silico using various computational softwares, such as AUTO￾DOCK, HEX.We are currently verifying our computational data

taking experimental approach, specifically the FRET.

OP-7

Mitochondrial electron transport chain

reactivity in the brain and eye tissues under

circadian rhythm alterations

M. B. Yerer1

, M. P. Alcolea Delgado2

, S. Aydog˘an1

,

F. J. Garcia-Palmer2 and P. R. Salom2

1

Faculty of Medicine, Department of Physiology, University of

Erciyes, 38039 Kayseri, Spain,2

Department of Biochemistry and

Molecular Biology, University of Balearic Islands, Mallorca,

Spain. E-mail: [email protected]

Mitochondria plays a central role in energy-generating processes

within the cell thorough the electron transport chain (ETC),

the primary function which is ATP synthesis via oxidative

Abstracts

43

phosphorylation (OXPHOS) which is shown to be related to age￾ing and apoptosis when this balance is destroyed under different

circumstances. This study is performed to investigate the effects

of alterations in the physiological melatonin levels via the circa￾dian rhythm changes, on the mitochondiral ETC in brain and

eye and how these changes are correlated to the pineal gland

melatonin receptor expressions. Fifty Sprague–Dawley male rats

weighing 200–250 g were used in five groups of different circa￾dian rhythms. The control group was 12/12 h of light/dark (L/D)

cycle. Different circadian rhythms of 24/0 h L/D, 0/24 h L/D,

16/8 h L/D and 8/16 h L/D cycles were applied to the groups for

1 week, respectively, in special cages where the duration of the

light and the climate can be adjusted. The melatonin receptors,

MEL1 and MEL2 expressions were determined by real-time PCR

in the pineal gland. The mitochondria of the brain and eye tis￾sues were isolated from the homogenates and the activation of

the mitochondrial OXPHOS complexes were determined by spec￾trophotometric micro-methods described before. Plasma melato￾nin levels were also determined by ELISA kit (IBL, Turkey).

Related to circadian rhythms, the plasma melatonin levels were

the highest in the 0/24 L/D group compared to the other groups

(p < 0.05) and the MEL1 and MEL2 receptor expressions were

also altered significantly by the circadian rhythms (p < 0.05).

The Complex I activity is found to be decreased significantly in

the 24/0 L/D group compared to the control and the 0/24 h L/D

group (p < 0.05) in the brain mitochondria whereas it was sig￾nificantly higher in the eye mitochondria compared to the control

(p < 0.05). Complex III activities were slightly lower in the 24/

0 h L/D group, whereas there was a significant increase in the

eye mitochondria in all the groups compared to the control

(p < 0.05). Furthermore, there was a significant increase in the

Complex IV and V activities in the brain mitochondria were sig￾nificantly higher 24/0 L/D group compared to its control

(p < 0.05) whereas they were found to be unaffected in the eye

mitochondria. As a consequence, this is the known first report to

show the MEL1 and MEL2 receptor expressions by real-time

PCR under different circadian rhythms. These alterations both in

the receptors and the plasma melatonin levels are found to be

correlated with the mitochondrial respiratory chain complexes

which are directly related to the energy metabolism in the cells

during the ageing process and the apoptosis. This study was

granted by NATO Science Fellowships A2 Programme of TUBI￾TAK.

Signaling and Cancer: Nuclear Receptor Connection

OP-8

Dysregulated Msx and Dlx gene expression in

epithelial odontogenic tumors

S. Ghoul-Mazgar1

, B. Ruhin2

, D. Hotton3 and A Berdal3

1

Laboratoire de Biologie Oro-Faciale et Pathologie, INSERM

U714-IFR-58, Universite´s Paris 7 and Paris 6 Laboratoire

d’Histologie-Embryologie, Faculte´ de Me´decine Dentaire de

Monastir, Tunisia,2

Stomatology and Maxillofacial Surgery

Department, Pitie´ Salpeˆtrie`re University Hospital, Paris Cedex 13,

France,3

Laboratoire de Biologie Oro-Faciale et Pathologie

INSERM U714-IFR-58, Universite´s Paris 7 and Paris 6, France.

E-mail: [email protected]

Odontogenic tumors are rare pathologies, mostly benign, located

in maxillary area. The most frequently observed benign epithelial

odontogenic tumors is called ameloblastoma and may although

give rise to the extremely rare malignant epithelial odontogenic

tumors, usually named odontogenic carcinomas.The differential

diagnosis between these tumors is so difficult regarding the

diverse clinical prognosis and therefore management. Homeodo￾main proteins comprise transcription factors that are essential in

many developmental processes. Homeodomain is encoded by a

highly conserved 60 amino acid sequence called homeobox that is

responsible for specific interactions with DNA. Non-clustered ho￾meobox genes are called non-HOX and include the Msx and Dlx

gene family. In this study, we examined the Dlx and Msx gene

expression by RT-PCR and in situ hybridization in recurrent 13

benign ameloblastomas and one malignant clear cell odontogenic

carcinoma (CCOC). Our data show specific expression pattern of

Msx and Dlx gene in the CCOC compared with benign amelobl￾astomas. Furthermore, exploring the expression pattern of signal

molecules by RT-PCR, Bmp2 was shown to be inactivated in the

carcinoma, but not Bmp4. Malignancy of epithelial odontogenic

carcinoma seems to be a multistep and highly heterogeneous pro￾cess requiring activation and deactivation of multiple and specific

genes suggesting exploration of homeogene expression to discrim￾inate benign ameloblastomas and odontogenic carcinomas.

OP-9

Ligand-specific dynamics of the androgen

receptor on its target promoter in living cells

T. I. Klokk1

, P. Kurys1

, C. Elbi2

, A. K. Nagaich2

,

A. Hendarwanto2

, T. Slagsvold1

, C. Y. Chang3

, G. L. Hager2

and F. Saatcioglu1

1

Department of Molecular Biosciences, University of Oslo, Oslo,

Norway,2

Laboratory of Receptor Biology and Gene Expression,

National Cancer Institute, Bethesda, USA,3

Department of

Pharmacology and Cancer Biology, Duke University Medical

Center, Durham, MD, USA. E-mail: [email protected]

Androgen receptor (AR) mediates the action of androgens, which

are important in the development and maintenance of the male

reproductive system and in pathologic conditions such as pros￾tate cancer. This is the basis for the routine use of antiandrogens

to block AR function in disease states, but little is known on the

mechanisms involved. We studied ligand-dependent AR interac￾tion with a target promoter in vivo, using photobleaching micros￾copy, kinetic modeling, FRET analysis, and in vitro chromatin

remodeling. The interaction of agonist-bound AR with the

MMTV promoter was rapid and transient. In the presence of

antagonist, and with a transcriptionally impaired AR mutant, an

even faster interaction was seen due to decreased residence time

on the promoter. The short residence times seen for AR in

response to all ligands support the ‘hit-and-run’ model and three￾dimensional genome-scanning hypothesis of transcription factor

action. Furthermore, agonist and partial antagonists, but not

pure antagonists, induced the recruitment of a chromatin-re￾modeling complex to the HRE. Finally, FRET analysis in vivo

demonstrated both intermolecular and intramolecular interac￾tions between the N- and C- termini of AR at the HRE. Thus,

three-dimensional scanning of the genome space, ligand-depend￾ent modulation of AR kinetic properties, recruitment of chroma￾tin remodeling complexes and proper intermolecular and

intramolecular interactions are all critical for the in vivo function

of AR on its target promoter.

Abstracts

44