Báo cáo khoa học: F1 – Structure and Function of Motor Proteins pot

We have reconstituted the sustained actin-based movement of a N-WASP- or formin- functionalized particle in a biochemically controlled medium, which enables measurement of force producti

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F1 — Structure and Function of Motor Proteins

F1-001

What single-molecule mechanics can tell us

about mitosis?

J Howard

Max Planck Institute of Molecular Cell Biology & Genetics,

Dresden, Germany E-mail: howard@mpi-cbg.de

Our laboratory is interested in the biochemical and biophysical

basis of cell structure The structure of a cell is determined

primarily by its cytoskeleton, which serves as a scaffold to support the plasma membrane, and as a network of tracks along which motor proteins transport sub cellular structures Our research is therefore focused on the mechanics of the cytoskeleton, with a particular emphasis on microtubules and microtubule-based motors On one hand, we are interested in the mechanisms by which these proteins work: i.c how do kinesins and dyneins convert chemical energy derived from the hydrolysis

of ATP into mechanical work used to move along or to

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depolymerize microtubules? And on the other hand, we are inter-

ested in the roles that microtubules and their motors play in cell

morphology and motility In this regard we are particularly inter-

ested in how the dynamic properties of microtubules and motors

drive spindle and chromosome movements in mitosis To address

these questions we are combining molecular biology techniques

with image processing, modeling, mechanical measurements and

single-molecule techniques

F1-002

Control of actin assembly in cell motility

M.-F Carlier

CNRS, Gif-sur-Yvette, France E-mail: carlier@lebs.cnrs-gif fr

Living cells change shape and move in response to environmen-

tal signals These motile processes play a pivotal role in mor-

phogenesis, migration of embryonic and metastatic cells,

angiogenesis, synaptic plasticity, immune response and interac-

tion of the host cells with pathogens They are generated by

polarized, spatially directed actin assembly It is the treadmilling

(dissipative turnover) of actin filaments, regulated by specific

proteins, which is responsible for force and directional move-

ment Two cellular machineries are responsible for spatially

directed initiation of actin filaments and they operate in distinct

processes, (i) the WASP-Arp2/3 system is at the origin of the

formation of a branched filament array; (ii) formins, in associ-

ation with profilin, catalyze the rapid processive assembly of

non-branched actin filaments We have combined a biochemical

and a biomimetic approach to understand the molecular mecha-

nisms of these auto-organized processes We have reconstituted

the sustained actin-based movement of a N-WASP- or formin-

functionalized particle in a biochemically controlled medium,

which enables measurement of force production in correlation

with structure and motility, and we can derive information on

the molecular mechanism of movement by single molecule

measurements

F1-003

The structure of the myosin VI motor reveals

the mechanism of directionality reversal

J Ménétrey!, A Bahloul!, C Yengo’, A Wells”, C Morris”,

H L Sweeney” and A Houdusse!

'UMRI44 - CNRS, Institut Curie, Paris, France, ? Departement of

Physiology, Pennsylvania School of Medicine, Philadelphia, PA

19104-6085 United States of America

E-mail: anne.houdusse@curie.fr

We have solved a 2.4 A structure of a truncated version of the

reverse direction myosin motor, myosin VI that contains the

motor domain and binding sites for two calmodulins Surpris-

ingly, the structure reveals only minor differences in the motor

domain as compared to plus-end directed myosins, with the

exception of two unique inserts The first insert is near the nuc-

leotide-binding pocket, and alters the rates of nucleotide associ-

ation and dissociation The second unique insert forms an

integral part of the myosin VI converter domain along with a

calmodulin bound to a previously unseen binding motif within

the insert This serves to redirect the effective “lever arm’ of

myosin VI, which includes a second myosin VI calmodulin bound

to an “IQ motif’, towards the pointed (—) end of the actin fil-

ament This repositioning largely accounts for the reverse direc-

tionality of this class of myosin motors We propose a model

incorporating a kinesin-like uncoupling/docking mechanism to

fully explain the movements of myosin VI

F1-004 Regulation and mechanics of myosin V

J R Sellers Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institute of Health, Bethesda, MD United States of America E-mail: sellersj@nhlbi.nih.gov

Myosin V is vesicle motor that moves processively on actin filaments

In melanocytes it functions in cooperation with microtubule motors

to localize melanosomes to the dendritic tips Myosin V dimerizes via a coiled coil motif in its tail region to produce a two-headed structure Each head is composed of a compact motor domain and a long neck formed by the association of calmodulin residues with six tandem IQ motifs This structure allows the molecule to take 36 nm steps along an actin filament corresponding to the helical pitch of the actin and, in turn, allows the molecule to keep its cargo positioned above the cytoskeleton We study the mechanics of the processive motor using optical trapping and total internal reflection fluorescence microscopy (TIRF) These data confirm that the neck of myosin V acts as a rigid lever arm and that myosin V moves in a hand- over-hand manner The enzymatic activity of myosin V is regulated

by calcium in vitro This is coupled to a large conformational change

in the molecule In the absence of calcium myosin V folds into a compact triangular-shaped structure in which the heads bend down and contact the globular tail domain Under these conditions the molecule migrates with a sedimentation coefficient of 14S In the presence of calcium or at high ionic strength the molecule opens up

to form T- or Y-shaped structures which sediment at 11S We propose that in the cell, the transition between the open, active state and the folded, inactive state is regulated by binding of receptor or docking proteins to the myosin V globular tail domain This would not only target the myosin to the proper cargo, but would also lock the myosin into an active form We are currently studying the interactions responsible for the folded, off state of this molecule

F1-005 F-actin modulates myosin conformational states by switch | loop movement

B Kintses!, M Gyimesi', W Zeng’, P B Conibear’,

C R Bagshaw and A Málnási-Csizmadia'

"Departement of Biochemistry, Eétvés Lordnd University, Buda- pest, Hungary, “Department of Biochemistry, University of Leices-

ter, Leicester, United Kingdom E-mail: amc26@leicester.le.uk

Myosin converts chemical energy to mechanical work It undergoes

a large conformational change during the ATPase cycle that results

a step on the actin filament The main question is how the functional regions communicate with each other Another problem concerns the development and relaxation of the mechanical strains during the catalytic cycle and their perturbation by external force

We have extensively characterized the energetic coupling between nucleotide binding, the so-called open/closed transition and actin binding using different single tryptophans located at the relay loop (W501, Dictyostelium sequence), the nucleotide binding region (W129) and the switch 1 region (W239 and W241) The fluorescence from native actin tryptophan residues is not significantly per- turbed on binding to myosin, although a fluorescence signal is detected as a consequence of a light scatter artifact ATP, ATPaS and ADP binding affected less than threefold by actin The isome- rization detected by W129 clearly precedes the dissociation of actin

in the case of ADP and ATP4S binding The fluorescence from the conserved W501 residue located at the distal end of the relay helix

is very sensitive to the lever arm disposition and the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre and post power-stroke conformations Actin modulates this equilibrium by no more than twofold These data suggest that actin activates another process in the mechanism,

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such as switch 1 movement, rather than influencing the switch 2

equilibrium Consequently, despite actin does not have major effect

on nucleotide binding and the equilibriums of the pre- and post-

power-stroke the fluxes of the kinetic routes change fundamentally

by actin binding: in the absence of actin phosphate release precedes

the closed-open transition while in ternary complex the main route

is that products releases follow the power stroke

F1-006

Calmodulin and calmodulin-like protein as

light chains for myosin-10: Specificity and role

in protein stabilization leading to prolonged

function

E E Strehler, A J Caride, A S Mauer and R D Bennett

Biochemistry and Molecular Biology, Mayo Clinic College of

Medicine, Rochester, MN, United States of America

E-mail: strehler.emanuel@mayo.edu

Vertebrate myosin-10 (MYO10) is involved in filopodial motility,

phagocytosis, and microtubule-F-actin interactions Each MYO10

heavy chain contains three IQ motifs, which are light chain binding

sites Previous work has shown that the first two IQ domains of

MYO10 bind calmodulin (CaM) whereas [Q3 alternatively binds

calmodulin-like protein (CLP) We performed stopped-flow experi-

ments with fluorescent derivatives of CaM (TA-CaM) and CLP

(TA-CLP) to analyze their binding to the IQ3 peptide TA-CaM

bound to IQ3 in the absence and in the presence of 100 M Ca? ”

In the absence of Ca** the binding was faster, albeit with lower

affinity, suggesting an even faster dissociation rate In the presence

of Ca?*, the time course of the TA-CaM-IQ3 reaction was best

described by an exponential function CLP was able to reduce the

amplitude of this exponential in the presence, but not in the

absence, of Ca** These results suggest a novel IQ domain interac-

tion, in which Ca** regulates binding of CaM to IQ3 by modula-

ting competition with CLP In HeLa cells over-expressing CLP,

endogenous MYO10 was strongly upregulated Likewise, transfec-

tion with GFP-MYO10 resulted in increased fluorescence in cells

that co-expressed CLP or excess CaM MYO10 upregulation resul-

ted in an increase in the size and number of filopodia Cells expres-

sing CLP displayed increased motility as indicated by their

shortened wound-healing time compared to control cells lacking

CLP CLP-dependent upregulation of MYOI10 was due to

increased protein stability CLP expression in specific epithelial

cells may thus prolong MYO10 function in conditions of elevated

intracellular Ca** and limiting CaM Acknowledgment: Suppor-

ted by grants from the Susan G Komen Breast Cancer Foundation

(EES) and the American Heart Association (AJC)

F1-007P

Crystal structure of the portal protein from

bacteriophage SPP1 and model for DNA

translocation

A A Lebedev!, M H Krause’, A Vagin', E V OrlovaŸ,

E J Dodson!, P Tavares* and A A Antson!

'YSBL, Chemistry Department, York University, York, United

Kingdom, ? Max-Planck Institut ftir Molekulare Genetik, Berlin,

Germany, * Birkbeck College, Department of Crystallography, Uni-

versity of London, London, United Kingdom, “Unité de Virologie

Moléculaire et Structurale, Gif-sur-Yvette, France

E-mail: fred@ysbl.york.ac.uk

The mechanism of DNA translocation into a viral procapsid

remains one of the most intriguing questions of viral particle

assembly [1] Tailed bacteriophages and herpes viruses have a specialized vertex for double-stranded DNA (dsDNA) entry into the procapsid during viral chromosome packaging [2, 3] The main component of this specific doorway is the portal protein, a circular oligomer with a central tunnel through which the DNA transfer occurs Together with viral ATPase (termi- nase) the portal protein forms a molecular motor that is able

to translocate DNA against high internal pressure [4] We determined the X-ray structure of the SPPI portal protein (gp6) in its 13-subunit oligomeric form where the tunnel residue segments, not visible in the previously determined structures of bacteriophage phi-29 portal protein [5, 6], are well defined and form a DNA-transfer arm Our X-ray and electron microscopy data suggest that DNA translocation is driven by a novel mechanism involving mechanical movements of arms along the inner walls of the tunnel We propose a model for DNA translocation where such movements propagate around the double helix of DNA similar to a “Mexican wave’? moving across a stadium

References

1 Hendrix RW Proc Natl Acad Sci USA 1978; 75: 4779-47783

2 Bazinet C, King, J Annu Rev Microbiol 1985; 39: 109-129

3 Droge A, Tavares P J Mol Biol 2000; 296: 103-115

4 Smith DE, et al Nature 2001; 413: 748-752

5 Simpson AA, et al Nature 2000; 408: 745-750

6 Guasch A, et al J Mol Biol 2002; 315: 663-676

F1-008P Unraveling the catalytic mechanism of the

bacteriophage T7 gene 4 helicase

D J Crampton, S Mukherjee, A van Oijen and

C C Richardson Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

E-mail: dcrampton@hms.harvard.edu The gene 4 helicase of bacteriophage T7 is a molecular motor that unwinds double-stranded DNA using the energy derived from the hydrolysis of deoxynucleoside 5-triphosphates Here

we present data pertaining to four essential activities of the helicase that act cooperatively to unwind DNA (i) Oligomer For- mation The T7 gene 4 helicase forms both heptamers and hexamers dependent upon the presence of nucleoside di- or triphosphates respectively We find that the heptamer cannot bind DNA but rather, in the presence of single-stranded DNA, heptamer converts to hexamer when both nucleoside di- and triphosphates are present together This conversion between oligomers is regulated by histidine 465 through differentiation

of the absence or presence of a y-phosphate on the bound nucleotide (ii) DNA Binding The loop comprised of residues 466—

475 has been postulated to be the major site for the binding of single-stranded DNA Lysines 471 and 473 of this loop were altered to determine their contribution to the overall binding of single-stranded DNA (iii) dTTP Hydrolysis We find that all subunits of the hexamer are active in the hydrolysis of dTTP Changing the catalytic base glutamate 343 to glutamine creates

a non-catalytic subunit that responds to dTTP in a manner similar to the effect of non-hydrolyzable analog 8,yy-methylene dTTP on wild-type helicase The presence of a single non-catalytic subunit per hexameric unit abolishes all dTTPase activity (iv) Translocation We are developing methods of observing movement along DNA by the gene 4 helicase using single-molecule fluorescence microscopy

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F1-009P

Myosin VI in chromaffin cells

M Dominik!, L Kilianekf, and M J Redowicz!

‘Laboratory of Cell Motility, Department of Muscle Biochemistry,

Nencki Institute of Experimental Biology, Warsaw, Poland,

?Laboratory of Confocal Microscopy, Nencki Institute of Experi-

mental Biology, Warsaw, Poland

E-mail: mdominik @nencki.gov.pl

Myosin VI (MVI), the member of one of at least 18 different

myosin families, is ubiquitously expressed in multicellular eukary-

otes It is an unusual actin-based motor since it walks towards

the minus end of actin filaments, in the opposite direction to

other myosins MVI is involved in subcellular transport of mem-

brane-containing structures, it has been found to be engaged in

clathrin-based endocytosis and cell spreading and migration

Here, we aimed at testing the involvement of MVI in chromaffin

granule trafficking within chromaffin cells MVI, but not myosin

V, has been found in vesicular fractions isolated from bovine

adrenal medulla and rat pheochromocytoma cells (PC12) MVI is

on the apical side of the vesicle and its association with the gran-

ule seems to be very tight as the stripping with high salt concen-

tration or high pH does not remove it from the granule surface;

MVI is stripped from the granules only after addition of Triton

X100 MVI colocalizes with dopamine-B-hydroxylase (DBH),

chromaffin granules marker, both in PC12 cells and primary cell

culture of adrenal medulla It has been observed that MVI and

DBH were still found on the same granules after their isolation

from PC12 cells, confirming strong association of MVI with the

granules Five minute stimulation of PC12 cells with 56 mM KCI

seems to affect MVI localization as the fluorescence intensity cor-

responding to MVI was enhanced within the perinuclear area

only in stimulated cells These preliminary data seem to indicate

the important role of MVI in secretory cells

F1-010P

Enzyme kinetics above denaturation

temperature

M Gyimesi, Z Simon and A Malnasi-Csizmadia

Department of Biochemistry, Eétvés Lorand University, Budapest,

Hungary E-mail: gyimesimate@cerberus.elte.hu

Wide range of temperature dependence of enzyme kinetics meas-

urements enables more precise determination of reaction energetics

and accurate separation of reaction steps We have developed a

novel temperature-jump/stopped flow method to measure enzyme

transient kinetics at high temperature even above denaturation

temperature of enzymes This method enables us to increase the

temperature in a millisecond time scale parallel with mixing of the

reactants Heat denaturation normally occurs in a second time

scale If the temperature is increased above the enzyme denatura-

tion temperature, essentially all of the events can be followed that

are faster than heat denaturation reaction We have tested the new

method on myosin ATPase and GFP folding/unfolding reactions

F1-011P

Changes of inter-subunit contacts in growing

filaments during salt-induced polymerization

of actin

A Galinska-Rakoezy, B Wawro and H Strzelecka-Golaszewska

Laboratory of Structural Muscle Proteins, Department of Muscle

Biochemistry, Nencki Institute of Experimental Biology, Warsaw,

Poland E-mail: galinska@nencki.goy.pl

Polymerization/depolymerization of actin underlies numerous

motile events in eukaryotic cells It is well established that

conformational changes in G-actin generated by exchange of its tightly bound Ca for Mg accelerate the nucleation reaction but

do not influence substantially the rate of filament growth during salt-induced polymerization of actin We used N, N”-1,4-phenyl- enebismaleimide (PBM) to probe the conformation of F-actin at various stages of filament growth during polymerization of CaATP-, MgATP-, and MgADP-G-actin Effects of the type of polymerizing salt, filament stabilization by phalloidin, and pro- teolytic cleavage of actin at Gly-42 within D-loop were also investigated The earlier described accumulation of ’upper dimer ’ (UD), a product of cross-linking neighboring protomers from two long-pitch F-actin strands, concomitant with disappearance

of initially formed “lower dimmers” (LD) with subunits in a non-filamentous, antiparallel orientation, was observed under all conditions except for a polymer of cleaved actin that required phalloidin binding to yield UD The increase in yield of UD closely followed filament assembly from MgATP-G-actin, whereas it lagged behind polymerization of CaATP- and MgADP-G-actin Phalloidin accelerated the filament assembly but did not eliminate the delay in UD formation These results, along with changes in morphology of growing filaments visualized by electron microscopy, suggest that the tightly bound Mg and initially bound ATP not only accelerate the nucleation reaction but also promote a rearrangement of F-actin structure that eliminates LD’s from growing filaments They also confirm the suggested role of D-loop in stabilization of both the longitudinal subdomain 2/1 contacts and of the cross-strand contacts in F-actin

F1-012P The dynein light chain binds to a non-coiled- coil tail domain of myosin-Va that includes an alternatively spliced exon coding for three

amino acid residues

Z Hédi!, A Németh', E Kovacs', C Hetényi!, A Bodor’,

A Perczel’ and L Nyitray!

‘Deptartment of Biochemistry, Eétvés Lordnd University, Buda- pest, Hungary, “Department of Organic Chemistry, Eétvos Lorand University, Budapest, Hungary E-mail: nyitray@cerberus.elte.hu Class V myosins are involved in short-range intracellular transport along actin filaments Of the three mammalian myosin-V heavy chain genes, mutations of MYOSA are responsible for the dilute phenotype and Griscelli syndrome typel in mice and humans respectively A dynein light chain (DLC) has been identified as a tail domain light chain of myosin-Va (myoSa) DLC may function as a cargo-binding and/or regulatory subunit of both motor proteins Our goal was to identify and characterize the binding site of DLC on myoSa Various fragments of myoSa tail and DLC were expressed in £ coli and human cells Forma- tion of their complex was analyzed by pull-down assays, gel fil- tration, and spectroscopic methods DLC was found to bind as a homodimer to a ~12 residues segment (Prol282-Thr1293) localized between the medial and distal coiled-coil predicted domains

of the tail The binding region contains 3 residues, coded by the alternatively spliced exon B that is essential for DLC binding Using CD spectroscopy, we demonstrate that binding of DLC to the intrinsically unstructured DLC binding domain (DBD) stabilizes the neighboring coiled-coil domain NMR spectroscopy and molecular docking simulations show that a short synthetic peptide of DBD binds to a surface groove on DLC, as has been found with other known binding partners of DLC We hypothes- ize that the second binding site of the DLC dimer is either occu- pied by the DBD of the other heavy chain, or by a cargo, or interacts with other regulatory proteins

Acknowledgment: Supported by OTKA T43746

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F1-013P

Mutagenic analysis of the HsdR motor subunit

of type IC restriction modification enzyme

EcoR124I

E Sisakova and M Weiserova

Laboratory of Molecular Genetics of Bacteria, Division of Cell

and Molecular Microbiology, Institute of Microbiology,

Czech Academy of Sciences, Prague, Czech Republic

E-mail: sisak@ biomed.cas.cz

Enzyme EcoR124I belongs to the IC family of restriction modifi-

cation enzymes, intelligent molecular motors It is able to detect

the methylation status of its DNA target sequence and respond

with alternative activities, methylation or translocation of DNA

While bound to its target site, it translocates DNA towards itself

simultaneously in both directions (500bp/sec) It uses the free

energy associated with ATP hydrolysis to translocate DNA so

that DNA cleavage occurs remote from the asymmetric recogni-

tion site The enzyme EcoR124I is multifunctional, multi-subunit

enzyme, composed of three different subunits, which are encoded

by the genes hsdR, hsdM and hsdS Products of all three genes

are required for DNA cleavage, producing the endonuclease

HsdR subunit is a multifunctional motor protein, which has been

shown to posses ATPase, helicase and restriction activity To

provide a fully functional molecular motor, which can never

cleave DNA, the amino acid motif X, the active site of the endo-

nuclease domain of the HsdR subunit, was subjected to site-

directed mutagenesis The complementation analysis proved that

the substitutions DISIA, E165A, E165D, E165H, K167A in the

HsdR subunit fully removed the restriction activity in vivo of the

EcoR124I enzyme The mutant subunits were separately overpro-

duced, purified and mixed with purified methylase to reconstitute

the EcoR124I endonuclease in vitro As a substrate for DNA

cleavage in vitro we used the plasmid pCFD30 containing a sin-

gle site for EcoR124I The test of restriction activity showed that

reconstituted endonucleases were not able to cleave covalently

closed plasmid DNA to linear DNA in contrast to the wild-type

enzyme

F1-014P

Dielectric and fluoroscopic study on the

dynamic effects of myosin-S1 with/without

ATP on the hyper-mobile water around actin

filaments

M Suzuki', M P Siddique!, T Miyazaki', J Mogami’,

E Katayama’, T Kodama’? and T Q P Uyeda’

‘Laboratory of Physicochemistry of Biomolecular Systems, Depart-

ment of Materials Science and Engineering, Tohoku University,

Sendai, Japan, * Division of Fine Morphology, The Institute of

Medical Science, The University of Tokyo, Tokyo, Japan,

*Molecular Enzymology, Department of Bioscience and Bioinfor-

matics, Kyushu Institute of Technology, Ihzuka, Japan, “Gene

Function Research Center, AIST, Tsukuba, Japan

E-mail: msuzuki@material.tohoku.ac jp

Using high resolution microwave dielectric spectroscopy, we have

recently shown hyper-mobile water molecules around actin fila-

ments (F-actin) which have a much higher rotational mobility

than that of bulk water [1] and its marked volume increase with-

out significant change in the ordinary hydration shell when the

myosin motor-domain (S1) binds to F-actin [2] Here, we report

that (i) hyper-mobile water around actin filaments has been

detected with the fluoroscopic technique based on the fact that

the fluorescence intensity of fluorophore molecules, such as ribo-

flavin and pyrene-derivatives in caotropic salt aqueous solutions

was in clear correlation with those viscosity B-coefficients, (ii) the fluorescence intensity from pyrenyl actin markedly increased when myosin-S1 binds to actin filaments, which is consistent with the dielectric result (BBRC, 2004), (iii) in the solution of acto-S1 fused chimera protein and skeletal actin copolymers the fluorescent intensity from pyrenyl-group (linked at Cys374 of actin SD-I) increased in the presence of ATP indicating a marked decrease of hyper-mobile water around actin filaments An important implication of the present result is that the $1/F-actin interaction forms an asymmetric field of viscosity-gradient along the filament axis, which drives the unidirectional sliding of S1 hydrolyzing ATP by means of thermal brownian movements References

1 Kabir SR et al Biophys J 2003; 85: 3154-3161

2 Suzuki M et al Biochem Biophys Res Comm 2004; 322: 340-

346

F1-015P Drosophila myosin V: solution kinetics and motile properties

J Toth'?, M Kovacs’, F Wang!, L Nyitray? and J R Sellers!

‘Laboratory of Molecular Physiology, NHLBI, National Institutes

of Health, Bethesda, MD, United States of America, *Biokémiai Tanszék, ELTE, Budapest, Hungary E-mail: tothj@mail.nih.gov Myosin V is the best characterized vesicle transporter myosin motor in vertebrates but it is unknown whether all members of the myosin V family share a common, evolutionarily conserved, mechanism To address this question, we performed steady state and transient kinetic measurements on a recombinant Drosophila myosin V SI fragment Interestingly, none of the product release steps can be identified as a single rate-limiting step The ADP release rate constant from acto-S1, which is the bottleneck in the vertebrate myosin V ATPase cycle, appears to be 5-fold higher than the maximal steady-state ATPase activity The Pi release rate constant from the acto-Sl-products complex is even faster, implying that Drosophila myosin V does not follow a myosin II-type kinetic pattern, either The key features of the ATPase cycle are the low ATP hydrolysis equilibrium constant (0.33) and the high steady-state actin attachment (96% at 5m actin) The behavior of double-headed Drosophila myosin V in an in vitro motility assay suggests that a single molecule of myosin V cannot take multiple steps on the actin filament while being attached to

it We propose a mechanism whereby the ATP hydrolysis and the so-called weak actin-binding equilibria define the steady-state ATPase rate rather than the product release process This mechanism suggests that Drosophila myosin V will not act as a single molecule vesicle transporter, in contrast to vertebrate myosin Vs However, it may be able to perform processive transport if present in small ensembles on the surface of its cargo

F1-016P

A novel centrosomal protein, Cep57 stabilizes

microtubules in vivo and binds FGF-2 in vitro

Y Yoshitake, N Tsuruoka and A Iwai Department of Biochemistry, Kanazawa Medical University, Uchi- nada, Ishikawa Japan E-mail: yositake@kanazawa-med.ac.jp

We have identified a gene encoding a novel protein that binds microtubules (MT) and fibroblast growth factor-2 (FGF-2) It was found as FGF-2 associated protein in the course of studying the function of the 24kDa isoform of FGF-2 in nuclei by yeast two-hybrid screening This gene shares a sequence similarity with

an uncharacterized cDNA reported in GenBank as KIAA0092 and the encoded protein is identical to Cep57, a coiled-coil

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pro-tein in the centrosomes The occurrence of Cep57 in human cen-

trosomes has been recently reported but its function is unknown

GFP-fused protein over-expressed in HeLa cells was predomin-

antly observed as filamentous structures and co-localized with

MT Weekly-expressed tagged proteins were co-localized with

MT as spindle, spindle pole and MT in the midbody of the mito-

tic cells and also localized in the centrosomes of interphase cells

Treatments disturbing the MT network did not have any effect

on this protein distribution in the cells Over-expressed Cep 57

protected the MT structure against those treatments Therefore,

Cep 57 stabilizes MT Recombinant Cep57 protein bound both

isoforms of FGF-2 (24kDa and 18kDa) in vitro However, the

localization patterns of endogenous Cep57 observed by immuno-

fluorescent staining using the antibodies differed in both patterns

of FGF-2 isoforms siRNA to Cep57 induced a morphological

change (spindle-like shape with a poor cytoskeletal MT network)

and decreased cell growth in HeLa cells Cep57 may work as a

MT stabilizing protein in the centrosomes and spindle pole, and

then play a role on MT network formation and cell division

F1-017P

A novel dynamic model for actin

polymerization based on the idea of

self-catalysis

J Zhang!” and Y L Wang?

‘Department Biology, Chongqing University of Medical Sciences,

Chongqing City, PR China, ?Key Lab of Biomechanics & Tissue

Engineering, Institute of Bioengineering, Chongqing University,

Chongqing City, PR China E-mail: zhangjunl017@sohu.com

More and more researches have recently demonstrated that there

exist multiple pathways in actin polymerization In order to eluci-

date the macroscopic dynamics of actin assembly, a novel phe- nomenological theory for actin polymerizing under simple thermodynamic conditions has been put forward The theoretical model, called self-catalytic dynamics, emphasizes that the process

of actin polymerizing can be thought of an autocatalytic chain reactions To set up the quantified kinetic equations group, three basic postulations is employed, which are in good agreement with the fundamental physical rules and published literatures: (i) the actin monomers driven by Brownian motion in solution can interact with each other, and further form actin dimer via non- covalent association under certain molecular colliding conditions; (ii) the formed actin dimer and higher polymer spontaneously own catalyzing ability and can catalyze free actin monomer or dimer changing into actin residues (that refer to the G-actin monomers existing in actin filaments); (iii) the polymerization/ depolymerization processes can go on synchronously at the both ends (generally, the barbed and pointed ends, sometimes, also at the side of the elongating filaments) with different polymerization/depolymerization rates According to the analyses and basic enzyme catalysis principles, a scheme of kinetic differential equations can be established with definite initial conditions By strug- gling out the integral curves of the equations group, we get a series of dynamic functions, via which we can accurately calculate the length distribution of filaments, the concentration of free G-actin and polymerized filaments versus the polymerizing time, and also quantificationally explain the treadmilling phenomenon

of actin turnover

F2-Single Molecule Biochemistry and Mechanics

F2-001

Recent Advances in Single Molecule

Biophysics

C Bustamante

Department of Physics, University of California, Berkeley, CA,

USA E-mail: carlos@alice.berkeley.edu

I will present our recent results on the packaging of DNA by the

connector motor at the base of the head of bacteriophage @29

As part of their infection cycle, many viruses must package their

newly replicated genomes inside a protein capsid to insure its

proper transport and delivery to other host cells Bacteriophage

@29 packages its 6.6mm long double-stranded DNA into a

42 nm dia x 54 nm high capsid via a portal complex that hydro-

lyses ATP This process is remarkable because entropic, electro-

static, and bending energies of the DNA must be overcome to

package the DNA to near-crystalline density We have used opti-

cal tweezers to pull on single DNA molecules as they are pack-

aged, thus demonstrating that the portal complex is a force

generating motor We find that this motor can work against

loads of up to 57 picoNewtons on average, making it one of the

strongest molecular motors ever reported Movements of over

5 mm are observed, indicating high processivity Pauses and slips

also occur, particularly at higher forces We establish the force—

velocity relationship of the motor and find that the rate-limiting

step of the motor’s cycle is force dependent even at low loads

Interestingly, the packaging rate decreases as the prohead is

filled, indicating that an internal pressure builds up due to DNA

compression We estimate that at the end of the packaging the capsid pressure is 6 MegaPascals, corresponding to an internal force of 50 pN acting on the motor The biological implications

of this internal pressure and the mechano-chemical efficiency of the engine are discussed

F2-002

The complete folding/unfolding trajectory

of a protein captured with single molecule force-clamp spectroscopy

J M Fernandez Biological Sciences, Columbia University, New York, NY 10027, USA E-mail: jfernandez@columbia.edu

A dense network of interconnected proteins and carbohydrates forms the complex mechanical scaffold of living tissues The recently developed technique of single molecule force spectroscopy has enabled a detailed analysis of the force-induced conformations of these molecules and the determinants of their mechanical stability These studies provide some of the basic knowledge required to understand the mechanical interactions that define all biological organisms The application of mechanical force to biological polymers produces conformations that are different than those that have been investigated by chemical or thermal denaturation, and are inaccessible to conventional methods of measurement such as NMR spectroscopy and X-ray crystallography Force-induced conformational transitions are physiologically

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important, and offer novel perspectives on the structure of bio-

molecules Recent developments in single molecule force spectros-

copy have enabled study of the full unfolding and refolding

pathways of a protein under force-clamp conditions Mechanical

unfolding of a protein placed under a constant stretching force

allows us to obtain precise information about the pathway and

kinetics of unfolding Force-quench experiments capture for the

first time the full folding pathway of a protein Due to the force

of gravity and the need of living organisms to perform mechan-

ical work, mechanical stretching is most likely to have played a

role in the evolution of proteins By contrast, the large changes

in temperature or chemical denaturants commonly employed in

protein folding studies are not found in living cells Hence, the

mechanical unfolding/folding trajectories captured by force-clamp

spectroscopy reflect much more closely the conformations and

pathways of proteins in vivo, compared to those obtained by

means of thermal or chemical manipulations This lecture will

focus mostly on the use of these novel techniques to study the

dynamic changes that proteins undergo in response to a mechan-

ical stretching force

References

1 Fernandez JM, Li HB Force-clamp spectroscopy monitors the

folding trajectory of a single protein Science 2004; 303: 1674—

1678

2 Carrion-Vazquez M, Li H, Lu H, Marszalek PE, Oberhauser

AF, Fernandez JM The mechanical stability of ubiquitin is

linkage dependent Nature Structural Biology 2003; 10(9): 738—

743

3 Li HB, Linke WA, Oberhauser AF, Carrion-Vazquez M,

Kerkvliet JG, Lu H, Marszalek PE, Fernandez JM Reverse

engineering of the giant muscle protein titin Nature 2002; 418:

998-1002

F2-003

Tethering single protein concatamers to

enable measurement of thermal noise

response by Atomic Force Microscopy (AFM)

K Byrne, M Kawakami, B Khatri and A Smith

Laboratory of Molecular and Nanoscale Physics, Department of

Physics and Astronomy, University of Leeds, Leeds,

West Yorkshire, UK E-mail: phykb2@phys-irc.leeds.ac.uk

The thermal noise response of single molecules under tension can

be measured using AFM and used to reveal both the conserva-

tive and dissipative response of the molecule This recently devel-

oped method has been applied successfully to single sugar

molecules tethered via non-specific interactions between the

AFM cantilever and the base substrate In order to apply the

method to single protein concatamers we have found that a more

durable means of tethering the molecules is required Single pro-

tein molecules can be suspended between a cantilever and sub-

strate using non-specific tethering but the tether breaks before

sufficient data can be captured to give an accurate noise spec-

trum in all cases observed thus far Tethering molecules through

covalent rather than non-specific bonds should enable longer dur-

ation experiments to be carried out with ease and has the added

advantage that the exact location of the tether point is known A

protocol to tether a modified 5-domain concatamer of [27

between a gold coated cantilever and gold substrate via distinct

covalent links is being developed The gold binding functionality

of the C-terminal cysteine residue and the ability of N-terminal

histadine residues to bind succinamide will be exploited The

lysine residues have been removed from the concatamer so that

succinamide-amine bonds should only form at the N-terminal

end of the concatamer A self assembled monolayer of the short

linker molecule Di-thio-bis-succinamidyl-propionate (DSP) on

gold will be used to tether the N-terminus The short linker will interfere only minimally in the thermal noise spectrum molecule

of interest

F2-004

Single molecule and single paritcle imaging

in solution and in live cells

X Zhuang Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

E-mail: zhuang@chemistry.harvard.edu Understanding the molecular mechanisms of complex biological processes is one of the major goals in modern biology As molecular and cell biology get increasingly quantitative, a com- prehensive understanding of biological processes at the molecular level is becoming more readily accessible However, roadblocks still exist, among which is the challenge that we face in character- izing the complex dynamics of biological processes The existence

of multiple kinetic paths and transient intermediate states often makes these processes difficult to dissect, as individual steps of a multi-step process are typically not synchronized among molecules To tackle this problem, we are exploring optical imaging techniques to monitor, in real-time, the behavior of individual biological molecules and complexes, in vitro and in live cells In this talk, I will report our recent progress in the following two areas (i) Molecular mechanisms of viral infection: our single- virus tracking experiments allow us to visualize the viral infection process in real time, dissect individual stages of the viral entry pathway, and obtain a better understanding of the molecular mechanisms governing the influenza infection (ii) Structural dynamics of RNA and ribonucleoprotein enzymes: our single- molecule studies provide critical insights into the molecular mechanisms governing RNA structural dynamics, and the effects

of proteins on the structural dynamics of RNA enzymes

F2-005

Single molecule analysis of protein

aggregation and prions by SIFT

U Bertsch!, K F Winklhofer, T HirschbergerẺ, J Bieschke!,

P Weber', F U Hartl’, P Tavan’, J Tatzelt”,

H A Kretzschmar! and A Giese!

'ZNP, University of Munich, Munich, Germany, ? Department of Cellular Biochemistry, Max-Planck-Institute for Biochemistry, Martinsried, Germany, *Department of BioMolecular Optics, University of Munich, Munich, Germany

E-mail: armin.giese@med.uni-muenchen.de Protein aggregation is a key event in a number of diseases such

as Alzheimer’s disease, Parkinson’s disease and prion diseases

We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT) In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes at the single particle level in complex mixtures containing different types of aggregates In the case of prion diseases the prion protein (PrPC), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie-isoform (PrPSc), which forms amyloid aggregates This conversion, which is crucial for disease progression, depends on direct PrPC/PrPSc interaction We developed a high-throughput SIFT assay for the identification of drugs, which interfere with this interaction at the molecular level Screening a library of 10000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by

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dose-response curves with half-maximal inhibitory effects ranging

from 0.3 to 60 um Among these, six compounds displayed an

inhibitory effect on PrPSc propagation in scrapie-infected N2a

cells Four of these candidate drugs share a N-benzylidene-ben-

zohydrazide (NBB) core microstructure Thus the combination of

high-throughput in vitro assay with the established cell culture

system provides a rapid and efficient method to identify new

anti-prion drugs, which corroborates that interaction of PrPC

and PrPSc is a crucial molecular step in the propagation of pri-

ons Moreover, SIFT-based screening may facilitate the search

for drugs against other diseases linked to protein aggregation

F2-006

Studying protein reaction kinetics by

fluorescence correlation spectroscopy in

microfluidic mixers

P Galajda!, J Puchalla”, R Riehn! and R H Austin!

' Department of Physics, Princeton University, Princeton, NJ,

USA, , “Department of Molecular Biology, Princeton University,

Princeton, NJ, USA E-mail: pgalajda@princeton.edu

Fluorescence correlation spectroscopy is a powerful method to

gather information about the motion and interactions of mole-

cules and particles in solution The autocorrelation of the fluores-

cence intensity fluctuation emerging from fluorophores traversing

a confined excitation/observation volume is used for analysis

Usually either diffusion or convection dominates on the time-

scales of the fluctuations Measurements lasts for seconds making

it inadequate for kinetic studies of most biomolecular reactions

Here we present a method to overcome this limitation Reactants

meet in a microfluidic diffusional mixer A steady-state flow

establishes a direct mapping of the temporal evolution of the

reaction to the spatial position along the channel opening a way

for kinetic studies The experimental conditions are set so that

we can extract both the characteristics of diffusion and flow from

the data A variety of biological reactions might be studied by

the above technique from protein folding to DNA-protein inter-

actions

F2-007P

Changes in cell morphology, viability,

proliferation and cytotoxicity associated with

helium-neon laser irradiation of diabetic

wounded human skin fibroblasts

H Abrahamse, N Houreld and D Hawkins

Laser Research Unit, Faculty of Health, University of

Johannesburg, Johannesburg, Gauteng South Africa

E-mail: heidi@twr.ac.za

Low Level Laser Therapy (LLLT) is a form of phototherapy

used to promote wound healing in different clinical conditions

Laser radiation has the capability to inhibit or stimulate cellular

activity in the absence of significant heating Currently, no uni-

versally accepted theory has explained the mechanism of laser

biostimulation Although not established, an alternative treat-

ment modality for diabetic wound healing includes LLLT Laser

biostimulation of such wounds may be of benefit to patients by

reducing healing time Structural, cellular and genetic events in

normal, wounded and unwounded diabetic induced human skin

fibroblasts was evaluated after exposing cells to increasing doses

of helium—neon (632.8 nm) laser irradiation Structural changes

were evaluated by assessing colony formation, haptotaxis and

chemotaxis Cellular changes were evaluated using cell viability,

(ATP), proliferation, (ALP), cytotoxicity (LDH) while the Comet

assay evaluated genetic integrity Morphologically, wounded

diabetic fibroblasts responded to single exposure of 5 J/em? with the highest rate of haptotaxis and chemotaxis indicating a stimu- latory effect while 16 J/cm? showed the lowest rate indicating an inhibitory effect Cell viability and proliferation changes indicated

a decrease at higher doses while a dose of 5.0 J/cm? appeared to stimulate mitochondrial activity, which leads to normalization of cell function with an increase in cell viability A dose of 5 J/em? showed the highest rate of cell viability and proliferation with a minimal amount of damage to the cell membrane or DNA, sup- porting morphological evidence In conclusion, LLLT of 5 J/em? stimulates migration, proliferation, and metabolism of wounded fibroblasts to accelerate wound closure

F2-008P Methionine sulfoxide reductase from class A: aminoacids involved in catalysis of the reductase step and in substrate binding

S Boschi-Muller, M Antoine, A Gand and G Branlant Laboratoire MAEM UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, vandoeuvre-lés-Nancy, France

E-mail: Sandrine Boschi@maem uhp-nancy.fr Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes that reduce protein-bound methionine sulfoxide back to Met in the presence of thioredoxine 7z vivo, the role of Msrs is described as essential in protecting cells against oxidative damages, and to play a role in infection of cells by pathogenic bacteria There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB MsrA is specific for the S epimer of MetSO, whereas MsrB reduces the R epimer Both Msrs present a similar catalytic mechanism of sulfoxide reduction by thiols via the sulfenic acid chemistry The rate of the reductase step that leads to the formation of the sulfenic acid intermediate is fast This sup- ports an activation of the catalytic Cys and an increase of the electrophilic character of the sulfur atom of the sulfoxide substrate via an acid catalyst from the active site Both activations should favor the efficiency of the reductase process Data will be presented which illustrate the role of some amino acids involved

in the catalytic mechanism of the reductase step and in the substrate recognition of MsrAs, specifically in the formation of the hydrophobic pocket responsible for the methyl group recognition

F2-009P Role of Induced cAMP Early Repressors in B/K

Gene Transcription

M.-H Choi, Y.-M Jang, Y.-S Jang and O.-J Kwon Department of Biochemistry, The Catholic University of Korea, College of Medicine, Seoul, South Korea

E-mail: cmh0912@hanmail.net B/K protein, structurally classified as a member of double C2 domain proteins, is highly expressed in PC12 cells We previously found that forskolin decreased the expression of both B/K mRNA and protein by PKA-dependent mechanism, and that inducible cAMP early repressor (ICER) might be involved in that process via the interaction with a CRE-like domain that is located in the promoter region of B/K gene Here, we showed the direct involvement of ICER in the mechanism of forskolin- induced down-regulation of B/K gene expression in PC12 cells

To investigate the role of ICER, we’ve successfully cloned four types of rat ICER (I, Ig, If and IIg) Transient transfection (24 h) of each construct resulted in the successful expression of ICERs, and the expression level was even higher than that induced by 3-h treatment of forskolin, the time when the expres-

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sion of ICER increased to the highest level In promoter assay,

ICER suppressed the promoter activity of B/K in CHO-K1 cells

that were cotransfected with the construct containing B/K CRE-

like sequence Inhibition level might not be related with the iso-

types of ICER but with the expression level in the cells, suggest-

ing the absence of isotype specificity of ICER in the repression of

B/K gene transcription Effect of ICER did not observed in the

cells cotransfected with the B/K construct having mutated CRE-

like sequence (AC:TG) Direct binding of B/K CRE-like

sequence to ICER was also observed in chromatin immunopre-

cipitation experiment These results strongly suggest that ICER

may be the principal suppressor molecule in the process of B/K

gene expression

F2-010P

Comparative structural and conformational

studies on two forms of beta lactoglobulin

(A and B) upon interaction with lead ton

A Divsalar and A A Saboury

Laboratory of Biophysical Chemistry, Institute of Biochemistry

and Biophysics, University of Tehran, Tehran, Iran

E-mail: divsalar@ ibb.ut.ac.ir

One of the most widely studied and fascinating proteins of animal

origin is Beta lactoglobulin (Blg) This polypeptide, with 150-170

amino acids (depending on the species), is an abundant compo-

nent of the milk of most animals, although it has not been detec-

ted in human milk Bovine Blg consists of 162 amino acid residues

and contains two disulfide bonds and a free thiol Its interaction

with a great variety of hydrophobic ligands has led to its inclusion

in the lipocalin (transport protein) superfamily Blg is a dimeric

native conformation at neutral pH, while the conformation at pH

2 is a monomeric but still native Thermodynamic studies have

been made on the effect of Pb’ on the structure of two forms of

Blg (A and B) in 50 mM sodium chloride at 27 °C using UV spec-

trophotometry, circular dichroism (CD) and fluorescence spectros-

copy UV spectrophotometry studies show that the protein

absorbance changes due to the increase concentration of ion lead

are a biphasic curve for Blg-A form and a monophasic sigmoid

curve for Blg-B form Far CD studies do not show any change on

the secondary structure of Blg-A upon interaction with concentra-

tions of 15.54 and 35.8 mm of Pb?*, while far CD studies show

considerable change in the secondary structure of Blg-B by

increasing the concentration of Pb** Near CD and fluorescence

spectroscopy studies show considerable change in the tertiary

structure of Blg-A related to the interaction with lead ion, but

these studies do not show any change in the tertiary structure of

Blg-B Different behaviors of Blg (A and B) upon interaction with

lead ion, related to different native structure of both proteins

F2-011P

Strong specific binding forces between

adhesive extracellular proteoglycan

carbohydrates

I Bucior', S Garcia-Manyes’, R Ros’, F Sanz’,

D Anselmetti*, M M Burger’ and X Fernandez-Busquets*

’ Friedrich Miescher-Institut, Basel, Switzerland, ? Department of

Physical Chemistry, University of Barcelona, Barcelona, Spain,

* Experimental Biophysics, University of Bielefeld, Bielefeld,

Germany, *Laboratory of Nanobioengineering, Barcelona Science

Park, University of Barcelona, Barcelona, Spain

E-mail: busquets@qf.ub.es

Specific carbohydrate-carbohydrate interactions are rarely repor-

ted in biologically relevant situations such as cell recognition

However, carbohydrate structures have immense structural diver- sity, a ubiquitous distribution in vertebrate and invertebrate tissues, and are associated with the cell surface, as required of cell recognition molecules Carbohydrate-carbohydrate interactions are characterized by relatively weak forces which, when multi- merized, can be easily potentiated by orders of magnitude, representing a highly versatile form of cell recognition and adhesion given the extraordinary plasticity of their structures Sponge cells associate in a species-specific process through multivalent interactions of carbohydrate structures on a type of extracellular bifunc- tional proteoglycan molecules termed spongicans In the marine sponge Microciona prolifera the spongican molecule, Microciona aggregation factor (MAF), self-interacts via a Ca’ *-dependent interaction We have dissected MAF into its components, tracking the individual self-binding units down to the circular core of the sunburst-like molecule and further down to a 200-kDa glycan (g200) Force spectroscopy data and surface plasmon resonance measurements reveal that the carbohydrate self adhesion is highly specific when compared with the binding to other sulfated carbohydrates such as chondroitin sulfate The strength of the binding per surface area between two spongican molecules is comparable

to that of focal contacts between vertebrate cells The results obtained reveal surprisingly high forces and selectivity for this most ancient cell adhesion system, and the existence of poly- morphism between ¢200 purified from different individuals, thus representing the first indication of specific carbohydrate—carbohydrate interactions implicated in the discrimination between cells

of different individual origin within a single species

Acknowledgments: This work was supported by grant BIO2002-00128 from the Ministerio de Ciencia y Tecnologia, Spain, that included FEDER funds

F2-012P Scanning probe microscopy as tool to study the stability of azurin in air

V Frascerra, G Maruccio, V Arima, L del Mercato,

P P Pompa, F Calabi, R Cingolani and R Rinaldi National Nanotechnology Laboratory of INFM, Ingegneria dell’ Innovazione, University of Lecce, Lecce, Italy

E-mail: vanessa.frascerra@unile.it Scanning probe microscopy (SPM) comprises a family of techniques that can be used as tool to study biomolecules morphology and properties on the atomic scale We focus on the use of the SPM to study the azurin stability in air Azurin is a metallo- protein involved in electron transfer during denitrification pathway of the bacterium Pseudomonas aeruginosa Thanks to its electron transfer properties, azurin and its physiological partners could be employed for the implementation of planar biosensors, with enhanced sensitivity and selectivity, exploiting a monolayer

of proteins that should act as an optimal surface for the analyte molecules to react with For this purpose it is fundamental that the immobilized proteins preserve their native conformation and properties, especially in ambient conditions First, Scanning Tunneling Microscopy (STM) allows us to achieve a very high resolution and to probe the electronic properties of Au (111)- immobilized Azurin, both in buffer solution and in air Our results demonstrates that immobilized proteins preserve their electron transfer function Secondly, the force distance mode of atomic force microscopy has been used to measure the interaction between tip and SiO2-immobilized azurin by mercaptosi- lanes The force curves show that the adhesion of the tip to the azurin is weaker than to the SiO2 and silanized surface The mechanical properties of azurin could be deduced by analyzing these curves and correlated with the native conformation of the protein

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F2-013P

Hydrogen peroxide mediates Rac1 activation

of S6K1

J.-W Han', Y.-G Chung!, S J Paek!, S H Park!, J.-S You',

J.-K Kang!, Y M Kim’, H Y Lee*, Y K Kim‘ and

H W Lee!

‘Lab of Biochem & Mol Biol, College of Pharmacy, Sungkyunk-

wan University, Suwon, Gyeonggi-do South Korea, “College of

Pharmacy, Duksungsung Women’s University, Seoul, South Korea,

*College of Medicine, Konyang University, Nonsan, Chungcheong-

nam-do South Korea, *College of Medicine, Kwandong University,

Gangneung, Gangwon-do South Korea

E-mail: jhhan551@skku.edu

We previously reported that hydrogen peroxide (H2O2) mediates

mitogen activation of ribosomal protein S6 kinase 1 (S6K1)

which plays an important role in cell proliferation and growth

In this study, we investigated a possible role of hydrogen perox-

ide as a molecular linker in Racl activation of S6K1 Overexpres-

sion of recombinant catalase in NIH-3T3 cells led to the drastic

inhibition of HO, production by PDGF, which was accompan-

ied by a decrease in S6K1 activity Similarly, PDGF activation of

S6K1 was significantly inhibited by transient transfection or sta-

ble transfection of the cells with a dominant-negative Racl

(RacIN17), while overexpression of constitutively active Racl

(RaclV12) in the cells led to an increase in basal activity of

S6K1 In addition, stable transfection of Rat2 cells with

RaclN17 dramatically attenuated the H,O, production by

PDGF as compared with that in the control cells In contrast,

Rat2 cells stably transfected with RaclV12 produced high level

of HO; in the absence of PDGF, comparable to that in the con-

trol cells stimulated with PDGF More importantly, elimination

of HO; produced in Rat2 cells overexpressing RaclV12 inhib-

ited the Racl1V12 activation of S6K1, indicating the possible role

of H,O> as a mediator in the activation of S6K1 by Racl How-

ever, H»O, could be also produced via other pathway which is

independent of Racl or PI3K, because in Rat2 cells stably trans-

fected with RaclN17, HO could be produced by arsenite which

has been shown to be a stimulator of H,O, production Taken

together, these results suggest that HO; plays a pivotal role as a

mediator in Racl activation of S6K1

F2-014P

Reaction of hydroxymethylarginine and

endogenous formaldehyde with

tetrahydrofolate producing N°,N'°-methylene

tetrahydrofolate coenzyme

L Hullán!, L Trézl”, Z M Jászay”, M Tejeda*, A Csiba”,

J Bariska® and T Szarvas’

"Department of Biochemistry, National Institute of Oncology,

Budapest, Hungary, *Department of Organic Chemical Technol-

ogy, Budapest University of Technology and Economics, Budapest,

Hungary, *Organic Chemical Technological Research Group, Hun-

garian Academy of Science, Budapest, Hungary, “Department of

Experimental Pharmacology, National Institute of Oncology,

Budapest, Hungary, ° Veterinary and Food Control Station, Buda-

pest, Hungary, Central Laboratory, National Institute of Rheuma-

tology and Physiotherapy, Budapest, Hungary, ‘Institute of

Isotopes Co Ltd., Budapest, Hungary E-mail: hullan@oncol.hu

The endogenous formaldehyde (E-CH,O) means the always exist-

ing CH,O content of various biological samples E-CH2O is sup-

posed to be bound to biomolecules, because it has no toxic

effect N°-hydroxymethyl derivatives of arginine (HMA) synthes-

ized by Trézl and co-workers from CHO and L-arginine may

belong to that type of molecules HMA was found in human blood and urine, and in plants (leaves, fruits and vegetables) in higher concentration than in human samples since HMA is one

of the products of photosynthesis We have proved that the reaction of HMA and THF produces — under physiological conditions — N°,N'°-methylene tetrahydrofolate (CH>THF), the coenzyme of thymidylate synthase (TS) In recent work 'C-HMA was synthesized and the reaction of '°C-HMA and THF was compared to that of °C-CH,O and THF by '3C-NMR

in the reaction mixture for producing of CH»-THF according to the Roberts-method which is used for the determination of TS activities The two reactions were also compared to the reaction between E-CH;O and THF To test the CH2-THF product measurement of TS activities was applied using either cytosol of P388 lymphoid leukemia tumor, or TS purified from Lactobacillus ca- sei Both the NMR spectra and the enzyme assays proved that the reactions produced CH,-THF, however, the rate of reaction

by free CH.O is higher than those of the two other reactions The results suggest that TS and probably other proteins are the most important molecules in keeping of E-CH,O in bound form Acknowledgment: This work was supported by National Sci- ence Foundation OTKA Grant No T034245

F2-015P Co-operation of H,0O.-mediated ERK activation with Smad pathway in TGF-61 induction of

p21WAF1/Cip1

Y K Kim’, E K Lee”, J W Park’, J Y Lee’, H Y Jung’,

H J Kim’, J H Park’, S.-N Kim’, H Y Lee*, H W Lee*

and J.-W Han?

"College of Medicine, Kwandong University, Gangneung, South Korea, College of Pharmacy, Sungkyunkwan University, Suwon, South Korea, *Skin Research Institute, Amore-Pacific Corporation, Yong-In, South Korea, *College of Medicine, Konyang University, Nonsan, South Korea E-mail: yksnbk @kwandong.ac.kr

Although it has been demonstrated that p21~4?’CP! could be induced by transforming growth factor-B1 (TGF-B1) in a Smad- dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood In this study, we investigated a possible role of hydrogen peroxide

(H,0>)-ERK pathway in TGF-B1 induction of p21¥47"P! in

human keratinocytes HaCaT cells Using pharmacological inhibi- tors specific for MAP kinase family members, we found that ERK, but not JNK or p38, is required for TGF-B1 induction of p21v4FVCr! ERK activation by TGF-B1 was significantly attenuated by treatment with N-acetyl-L-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF-

Bl, mainly H,O., stimulates ERK signaling pathway to induce the p21¥4F VCP! expression In support of this, TGF-f1 stimulation caused an increase in intracellular ROS level, which was completely abolished by pre-treatment with catalase ERK activation does not appear to be associated with nuclear translocation

of Smad-3, because ERK inhibition did not affect nuclear translocation of Smads by TGF-Bl, and H,O, treatment alone did not cause nuclear translocation of Smad-3 On the other hand, ERK inhibition led to the disruption of interaction between Smad-3 and Spl induced by TGF-B1, indicating that ERK signaling pathway might be necessary for their interaction Taken together, these results suggest that activation of H,O2-mediated ERK signaling pathway is required for p21¥4"/CP! expression

by TGF-B1 and led us to propose a co-operative model whereby TGF-Bl-induced receptor activation stimulates not only a Smad pathway but also a parallel H,O2-mediated ERK pathway that acts as a key determinant for association between Smads and Spl transcription factor

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