Sociedad Argentina de Protozoologa 02-05 de Noviembre de 2008

nucleoside diphosphate kinases

Claudio A. Pereira
Laboratorio de Biologa Molecular de Trypanosoma cruzi Instituto de Investigaciones Medicas A. Lanari Universidad de Buenos Aires - CONICET

Trypanosoma cruzi yp

Research Topics
Argininekinase Arginine kinase arginine+ATPphosphoarginine+ADP

Adenylatekinase 2ADPATP+AMP

Nucleoside diphosphate kinase N1TP + N2DP N1DP + N2TP

Reasons t study phosphotranspherases: R to t d h h t h

1. There 1 Th are many phosphotransferase of trypanosomatids absent h h t f ft tid b t in mammals. 2. 2 Are involved in processes that require a rapid energy source or nucleotide interconversion 3. There is little information about the energy flow in some structures such as flagella and nucleus 4. The movement of ATP within a cell is not completely clarified p y

NDPK functions
Far more than HOUSEKEEPING ENZYMES, other than the biosynthetic processes: bi h i
1. 2.

Signal transduction by providing GTP to G proteins activation. Signal transduction by transference of the phosphohistidine phosphate to different acceptors as part of a phosphorylation cascade. It was also involved in bacterial two-component signal tr n du ti n systems. t m n nt ign l transduction t m Several functions in cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility were attributed attributed. DNA processing: in humans NM23-H2/NDPK B is a transcription factor with sequence-specific DNA binding activity. Recognition, activity Recognition cleavage and structural alterations of the DNA targets suggest a possible participation in DNA repair.

3.

4.

NDPKs background
1995

TcNDPK1?

Nuclear TbNDPK1

T T yps TriTryps
DATA MINING:

TcNDPK1: canonical short isoform short TcNDPK2: DM10-containing long isoform TcNDPK3: T NDPK3 putative (id T NDPK2) i (idem TcNDPK2) TcNDPK4: putative atypical long isoform

NDPK de T. cruzi: first ap Ks : pproach h

TcND DPK1: a novel functio l ion

T TcNDP alig PKs ignemnt nts

TcN cNDPK1 Crystal 1 ls

Other results... results

BZL res sistance

TcNDPK2: What is the role of DM10 motifs?

A novel Trypanosoma cruzi nucleoside diphosphate kinase targeted to the flagellum
Mariana R. Miranda, Len A. Bouvier, Gaspar E. Canepa and Claudio A. Pereira*

Laboratorio de Biologa Molecular de Trypanosoma cruzi (LBMTC), Instituto de Investigaciones Mdicas Alfredo Lanari, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina.

DM10: Domains in hypothetical proteins in Drosophila, C. elegans and mammals. Occurs singly in some nucleoside diphosphate kinases. NDPK2

Dig gitonin e extraction on

DM10 alignme ent

E-value: 4e

-08

DM10 TcNDPK2 DM10-TcNDPK2 smart00676

6 RYAFKVDYYDPQA--NLTRQYLLLYYTEDATIEMHDLKTK------RVFLKRCAYPS---------LGVQELFIGATVN RYAFKVDYYDPQA NLTRQYLLLYYTEDATIEMHDLKTK RVFLKRCAYPS LGVQELFIGATVN 4 VLRFDAYWDDPVAmpGLIRRFKISYYLEDDTIEIFEPDVRnsgilgGTFLRRQRVPKpsppddpefYTASDLNVGTTIN

DM10-TcNDPK2 69 FSRSLKLVDYgDEATRQ 85 smart00676 84 FGRQFRIYDC-DEFTRN 99

NDPK3 E-value: 3e
-07

DM10-TcNDPK3 smart00676

9 RLALYCERQDDISQ--LVRRFVLFFFYEDRSIEMRE-IPKNV-----LYLRRAPFP---------HLKKDDFTLGASLTI 4 VLRFDAYWDDPVAMpgLIRRFKISYYLEDDTIEIFEpDVRNSgilggTFLRRQRVPkpsppddpeFYTASDLNVGTTINV

DM10-TcNDPK3 72 NGGIVKITDYaDEVTRVLCEKKS 94 smart00676 84 FGRQFRIYDC-DEFTRNYLESKG 105

NDPK2 vs NDPK3 Expect = 3e-13 Identities = 25/79 (31%), Positives = 45/79 (56%)

TcNDPK2 TcNDPK3 TcNDPK2 TcNDPK3

6 9 66 69

RYAFKVDYYDPQANLTRQYLLLYYTEDATIEMHDLKTKRVFLKRCAYPSLGVQELFIGAT RLALYCERQDDISQLVRRFVLFFFYEDRSIEMREIPKNVLYLRRAPFPHLKKDDFTLGAS VNIFSRSLKLVDYGDEATR LTINGGIVKITDYADEVTR 84 87

65 68

DM10 motifs participate in the TcNDPK localization

TcNDPK2 (WT, CE) TcNDPK2 (WT, WC)

GFP (DM10-GFP, CE)

PAR2 (WT, CE)

PAR-2

TcNDPK2

TcNDPK2 regulation mechanisms

Glycosidases

Phosphatases

Trans-splice mapping

NO

NO

Additional Data

TcNDPK2 TcNDPK3

Future work work...

Explore the interrelations of different phosphotransferases in the cell Validation of the different TcNDPK isoforms Addressing the functional aspects of TcNDPK1 in relation with its nuclease activity, BZL resistance and TcNDPK2 in flagella Study the stage-specific and other regulation factors

AUTHOR AUTH RS & Co o.

LAB MEMBERS
Mariana Miranda (NDPKs)

Labora Labora atorio de Biologa Molecu de atorio de Biologa Molecu de e e a a cular cular Trypa Trypa anosoma cruzi anosoma cruzi a a

Leon Bouvier (ADKs) Gaspar Canepa (Transporters) Martn Vzquez (FCEyN,UBA) Carolina Carrillo (Instituto Leloir) Esteban Serra (IBR, Rosario) Antonio Uttaro (IBR, Rosario) Carlos Aguilar (UNSL, San Luis) International collaborations Michael Ginger (Lancaster University) SUPPORT ANPCyT: PICT RED 0300, PICT 33431 CONICET: PIP RED 5492 UBA: UBACyT X073
NE ETWORK WO ORK GROUP P

Institu de In Institu de In uto uto nvestigac nvestigac ciones M ciones M Medicas Medicas

National ll b N i l collaborations i

IDIM (C I I IDIM (C CONICE -UBA) CONICE UBA) ET- A) ET A)