Para vivir un buen noviazgo

due to their multiple and vital functions, stress proteins play a fundamental role in the pathology of several human diseases. aberrantly high levels of certain HsP classes are characteristic in cancer cells and the converse situation applies for type-2 diabetes or neurodegeneration. In accordance, understanding the mechanism whereby mammalian cells can elicit a stress protein response is of key importance. In ad- dition, correcting the defects of membrane domains, engaged in the generation and transmission of stress signals may be of paramount importance for the de- sign of new drugs with the ability to induce or attenu- ate the level of a particular class of heat shock proteins (see fig.2.).

Figure 2. Membrane-mediated stress protein response and the cellular localization of HSPs (highlighted by HSP70) in mammalian cells (see Horváth et al, BBA, 2008). HSF1 is a key coordinator of the

initiation of heat shock gene transcription, which is activated mainly by the appearance of denatured or misfolded proteins. In addition, stress sensing-signaling mechanisms operate through stress-induced mem-

BIOC

H

EM

IS

TR

brane rearrangements. Such typical membrane-mediated changes that are evidenced to refine the expression of heat shock genes are the non- specific clustering of the growth factor receptors associated with mem- brane microdomains (“rafts”) (1) or the activation of phospholipases (2), which sequester themselves into unsaturated-rich microdomains and cleave arachidonic acid, a known HSP inducer. Stress activation of such pathways alters the nuclear accumulation and transactivation capacity of HSF1 (3) via its covalent post-translational modifications and ultimate- ly retailor the abundance and profile of HSPs. The function of individual HSPs (highlighted on the scheme by HSP70) depends on their intracel- lular, membrane bound or extracellular location. The major action of chaperone proteins in the cytosol is to maintain protein homeostasis (4). HSP70 can promote cell survival by inhibiting lysosomal membrane per- meabilization via the interaction with specific lipids (5). We are currently

studying the interaction of HSPs with cellular lipid droplets. Experimen- tal evidence is accumulating in favor of the presence of HSP70 (and other HSPs) in lipid rafts as components of signaling or trafficking platforms (6). HSPs can also associate with specific lipids and proteins in the plasma membrane, inducing “membrane stabilization” and/or exhibiting an im- munogenic potential (7). HSP70s of extracellular location (8) have immu- nomodulatory capacities and are potent agents in the activation of the innate and adaptive immune system.

Contact: [email protected]

BIOC

H

EM

IS

TR

the group carries out its research activity in both the Biochemistry and the enzimology Institutes.

an ever-increasing number of diseases have been shown to originate from protein misfolding. among them, there are more than 20 diseases [including alzheimer's, Parkinson's, Huntington's and trans- missible spongiform encephalopathies (tses)] that are characterized by amyloidal protein deposits and thought to share common structural features and mechanism of action. (the amyloidal deposits usu- ally consist of fibres that contain misfolded proteins with a β-sheet conformation.) tses, which we chose as a model for the study of these conformational diseases, have drawn special attention due to the outbreak of a new variant of creutzfeldt-Jakob dis- ease (nvcJd) in the united Kingdom that seemed to be associated with eating beef from tse-infected cattle.

tse in humans may result from infection (nvcJd, iatrogenic cJd, kuru); can be inherited when a germ line mutation exists in the PrnP gene that encodes

the prion protein [familial cJd, Gerstmannstreüssler- scheinker syndrome (Gsss), fatal familial Insom- nia]; and can be sporadic (sporadic cJd) when the origin of the disease is neither determined nor un- derstood. tses are also observed in other mammals including sheep, goats, deer, elk, cattle, mink, cats and zoo animals.

The primary symptoms of tses usually include progressive dementia and ataxia, associated with spongiform degeneration of the brain and accumu- lation of an abnormal protease-resistant form of the prion protein (PrPres_{) in the central nervous system.}

(The normal, cellular form of this protein is denoted as PrPc_{.) after symptoms are first manifested, the}

progression of the disease can be dramatically rapid, leading inevitably to the death of the affected indi- vidual.

PrPc _{is a mainly α-helical, protease-sensitive, sol-}

uble monomer protein with a disordered n-terminus (approximate residues 23–127) and a structured c- terminal domain (residues 128–228, figure 1.).

Transmissible spongiform encephalopathies (TSEs) are deadly neuro-degenerative disorders among humans and mammals. The infectiousness is the most alarming feature of these diseases. It can be transmitted to humans from animals through the food chain and from humans by blood or organ donation or by infected medical devices. There is no cure or sufficient sensitive early detection for TSEs. The infectious agent, called prion, is believed to be a normal cell protein, the prion protein, with an unknown conformation that is different from its normal confor- mation. Our aim is to understand the conformational transition of the prion protein to disease-associated forms at the molecular level by combining the results of biochemical and biophysical measurements, cell-free conversion reactions, and experiments in cell and animal model systems.

ConfoRmaTIonal DIseases GRoUp

(membRane anD sTRess bIoloGY UnIT)

Ervin WELKER / Principal Investigator, Group Leader

Frida FODOR / staff scientist Adrien BORSY / staff scientist Antal NYESTE / Phd student Eszter TóTH / Phd student Krisztina HUSZáR / Phd student Petra BENCSURA / Phd student Judit BAUHOCH / technician Erika ZUKIC / technician

BIOC

H

EM

IS

TR

Figure 1. PrP is a 208 amino-acid residue glycosylated protein, which is

connected to the cell surface by a glycosyl phosphatidyl inositol (GPI) an- chor. However, only 110 residues (121-231) of the PrP domain are shown here. The two glycosylation sites are Asn 181 and Asn 197, which are both shown in yellow. One disulfide bond is present in the protein (colored gold here) connecting the second and third helices via Cys179-Cys214. The protein is colored by secondary structure. Alpha helices=magenta, beta sheets=blue.

By contrast, PrPres _{has a very compact structure,}

which is high in both α and β conformations, exists as oligomers and can be dissolved only by denaturing in GdnHcl or detergents. The disease-associated protein (PrPres_{) has partial protease-resistance; its n-termi-}

nal ~6 kda fragment is digested under conditions in which the highly compact c-terminal domain remains

intact. Both forms of the protein contain a single di- sulfide bond and two glycosylation sites. The generally accepted assertion that the difference between the cel- lular and disease-associated forms of the prion protein is purely conformational with respect to its disulfide bond patterns is based on our earlier experiments.

There is no cure for the disease, and early diagnosis is not available to discriminate between tse-infected unsymptomatic and uninfected individuals. such a sensitive, rapid, economical and non-invasive screen- ing test is vital with respect to animals destined for the human food chain, and to humans, who may partici- pate in tissue and blood donation programs. as with any other disease, a detailed mechanistic understand- ing of pathogenesis is the most effective approach for the development of sensitive predictive diagnostic and efficacious therapeutic regimens.

our aim is to understand tses at molecular and cellular levels as well as at the level of the whole organ- ism as a model for the formation of amyloid fibrils in conformational diseases.

Contact: [email protected]

BIOC

H

EM

IS

TR

Antal Kiss

Heitman, J., Ivanenko, t. and Kiss, a. (1999). dna nicks in- flicted by restriction endonucleases are repaired by a reca- and recB-dependent pathway in Escherichia coli. Mol. Micro-

biol. 33: 1141-1151.

raskó, t., finta, c. and Kiss, a. (2000). dna bending induced by dna (cytosine-5) methyltransferases. Nucleic Acids Res. 28: 3083-3091.

simoncsits, a., tjörnhammar, m.-L., raskó, t., Kiss, a. and Pongor, s. (2001). covalent joining of the subunits of a ho- modimeric type II restriction endonuclease: single-chain PvuII endonuclease. J. Mol. Biol. 309: 89-97.

Kiss, a., Pósfai, G., Zsurka, G., raskó, t. and venetianer, P. (2001). role of dna minor groove interactions in substrate recognition by the m.sinI and m.ecorII dna (cytosine-5) methyltransferases. Nucleic Acids Res. 29: 3188-3194.

roberts, r.J., Belfort, m., Bestor, t., Bhagwat, a.s., Bickle, t.a., Bitinaite, J., Blumenthal, r.m., degtyarev, s.K., dryden, d.t.f., dybvig, K., firman, K., Gromova, e.s., Gumport, r.I., Halford, s.e., Hattman, s., Heitman, J., Hornby, d.P., Janu- laitis, a., Jeltsch, a., Josephsen, J., Kiss, a., Klaenhammer, t.r., Kobayashi, I., Kong, H., Krüger, d.H., Lacks, s., marinus, m.G., miyahara, m., morgan, r.d., murray, n.e., nagaraja, v., Piekarowicz, a., Pingoud, a., raleigh, e., rao, d.n., re- ich, n., repin, v.e., selker, e.u., shaw, P.-c., stein, d.c., stod- dard, B.L., szybalski, W., trautner, t.a., van etten, J.L., vitor, J.m.B., Wilson, G.G. and Xu, s. (2003). a nomenclature for re- striction enzymes, dna methyltransferases, homing endonu- cleases and their genes. Nucleic Acids Res. 31: 1805-1812. tímár, e., Groma, G., Kiss, a. and venetianer, P. (2004). chang- ing the recognition specificity of a dna-methyltransferase by

in vitro evolution. Nucleic Acids Res. 32: 3898-3903.

rathert, P., raskó, t., roth, m., Ślaska-Kiss, K., Pingoud, a., Kiss, a. and Jeltsch, a. (2007). reversible inactivation of the cG-specific sssI dna-(cytosine-c5)-methyltransferase with a photocleavable protection group. ChemBioChem 8: 202-207. chuluunbaatar, t., Ivanenko-Johnston, t., fuxreiter, m., me- leshko, r., raskó, t., simon, I., Heitman, J. and Kiss, a. (2007). an ecorI-rsrI chimeric restriction endonuclease retains pa- rental sequence specificity. BBA-Proteins Proteom. 1774: 583- 594.

Kiss, a. and Weinhold, e. (2008). functional reassembly of split enzymes on-site: a novel approach for highly sequence- specific targeted dna methylation. ChemBioChem 9: 351-353. van der Gun, B.t.f., Wasserkort, r., monami, m., Jeltsch, a., raskó, t., Ślaska-Kiss, K., cortese, r., rots, m.G., de Leij, L.f.m.H., ruiters, m.H.J., Kiss, a., Weinhold, e. and mcLaugh- lin, P.m.J. (2008). Persistent down-regulation of the pancarci- noma-associated epithelial cell adhesion molecule via active intranuclear methylation. Int. J. Cancer 123: 484-489.

Kiss, a., Balikó, G., csorba, a., chuluunbaatar, t., medzi- hradszky, K.f. and alföldi, L. (2008). cloning and charac- terization of the dna region responsible for megacin a-216 production in Bacillus megaterium 216. J. Bacteriol. 190: 6448-6457.

tímár, e., venetianer, P. and Kiss, a. (2008). In vivo dna protection by relaxed-specificity sinI dna methyltransferase variants. J. Bacteriol. 190: 8003-8008.