Das Kostmann-Syndrom ist eine rezessive Form schwerer kongenitaler Neutropenie, ein ange-borener Immundefekt. Es zeichnet sich durch eine Verminderung der Anzahl neutrophiler Granulozyten im peripheren Blut (<500/µl) der Patienten aus. Dem Kostmann-Syndrom liegen ursächlich nonsense-Mutationen im Gen HAX1 (HS-1 associated protein X-1) zugrunde, weshalb die Patienten an HAX1-Defizienz leiden, die schon im frühen Säuglingsalter zu wied-erkehrenden bakteriellen Infektionen führt.
In der Literatur wird eine Lokalisation von HAX1 sowohl in den Mitochondrien als auch im endoplasmatischen Retikulum, Nukleus und Zytoplasma angegeben. Um die teils widersprüch-lichen Aussagen zu spezifizieren, stand im Vordergrund der vorliegenden Doktorarbeit die sub-zelluläre Lokalisation von HAX1.
Für die Untersuchungen von HAX1 wurde ein Expressionskonstrukt generiert, mit dem Fibro-blasten und HeLa-Zellen mittels retroviralen Gentransfers transfiziert wurden. Die Zellen wur-den mit speziellen Zellorganellmarkern für Mitochondrium, endoplasmatisches Retikulum, Golgi-Apparat, Lysosom, Zytoskelett und Nukleus gefärbt und mittels Fluoreszenz- und 2-Photonen-Mikroskopie analysiert. In funktionellen Studien wurde daraufhin das Verhalten des Proteins in Bezug auf seine Lokalisation unter verschiedenen apoptotischen Bedingungen (FCS-Deprivation, Staurosporin-, Doxorubicin- und H2O2-Behandlung) beobachtet. Die Rolle von HAX1 in eine zelluläre Signalkaskade wurde schließlich exemplarisch an der Funktion des TLR3 (toll-like receptor 3) durch einen IFN-β-ELISA getestet.
Die mikroskopischen Ergebnisse zeigten insbesondere in der präziseren 2-Photonen-Mikroskopie eine überwiegende Lokalisation von HAX1 in den Mitochondrien. Dort wird durch Cytochrom c-Ausschüttung der intrinsische Weg der Apoptose eingeleitet. Die mitochon-driale Lokalisation von HAX1 bestätigt dessen proklamierte antiapoptotische Funktion. Der durch die HAX1-Defizienz bedingte Ausreifungsstopp im Promyelozyten-/Myelozyten-Stadium der Neutrophilen ist somit auf die erhöhte Apoptoserate der Zellen zurückzuführen.
Nach Apoptosestimulation in HAX1-transfizierten Zellen kam es zu keiner Translokation des Proteins aus den Mitochondrien zum Nukleus. Deshalb wurde die These, dass HAX1 als Tran-skriptionsfaktor im Zellkern fungiert, verworfen. Es gab zudem keine Hinweise dafür, dass HAX1 bei der Aktivierung von TLR3 in dessen Signalkaskade involviert ist.
In Zukunft sind weitere Studien zur Klärung der exakten molekularen Mechanismen bei HAX1-Defizienz nötig. Insbesondere ist dabei die antiapoptotische Funktion von HAX1 in den Mito-chondrien genauer zu untersuchen. Weitere Ansatzpunkte zur Klärung der komplexen, multi-funktionellen Rolle von HAX1 bietet die Forschung über die Funktionen von HAX1 bei der Aufrechterhaltung des mitochondrialen Membranpotentials, der Signaltransduktion und in der Kontrolle des Zytoskeletts.
6. ABKÜRZUNGEN
AEBSF - 4-(2-Aminoethyl)-Benzensulfonylfluorid AIF - apoptosis inducing factor
AML - Akute myeloische Leukämie ANC - absolute neutrophil count AP-1 - activator protein 1
Apaf-1 - apoptotic protease activating factor 1 APS - Ammoniumpersulfat
ARCM - adult rat cardiac myocyte Arp - actin related protein
Bcl-2 - B-cell lymphoma-2
BHRF1 - BamHI fragment H rightward open reading frame 1 bp - Basenpaare
BSA - Bovines Serumalbumin BSEP - bile salt export protein CD - clusters of differentiation
cDNA - komplementäre Desoxyribonukleinsäure CFU - colony forming unit
DAPI - 4’,6-Diamidin-2’-Phenylindol-Dihydrochlorid DED - death effector domain
dH2O - deionisiertes Wasser
DIABLO - direct IAP-binding protein with low pI DISC - death inducing signaling complex
DMEM - Dulbecco’s Modified Eagle Medium DMSO - Dimethylsulfoxid
dNTP - Desoxyribonucleosidtriphosphat dsRNA - Doppelstrang-RNA
DTT - Dithiothreitol
EBNA-LP - Epstein-Barr virus nuclear antigen leader protein
EBV - Epstein-Barr Virus
ECL - verstärkte Chemilumineszenz (enhanced chemiluminescence) EDTA - Ethylendiamintetraessigsäure
ELA2 - Elastase 2
ELISA - Enzym gekoppelter Immunnachweis (enzyme linked immunosorbent assay) Endo G - Endonuclease G
ER - Endoplasmatisches Retikulum FACS - fluorescence activated cell sorting FADD - Fas associated death domain FCS - fetal calf serum
FITC - Fluoreszeinisothiocyanat FSC - forward scatter
GAPDH - Glycerinaldehyd-3-phosphat-Dehydrogenase G-CSF - granulocyte colony-stimulating factor
GFI-1 - growth factor independent 1 GST - Glutathion S-Transferase HA - Hämagglutinin
HAX1 - HS-1 associated protein X-1 HIV - human immunodeficiency virus HLA - human leukocyte antigen
HRP - Meerrettichperoxidase (horseradished peroxidase) HS1 - haematopoietic lineage cell-specific protein 1 HSV1 - Herpes simplex Virus 1
HtrA2 - high temperature requirement A2 IAP - inhibitory apoptosis proteins IL-1RII - Interleukin-1 Rezeptor Typ II IRES - Interne Ribosomale Eintrittsstelle IRF-3 - interferon regulatory factor
IU - Internationale Einheit (international unit)
kb - Kilobasen kDa - Kilo Dalton
LAMP 3- lysosomal associated membrane protein 3 LEF-1 - lymphoid enhancer-binding factor 1 LMP-1 - latent membrane protein 1
LP - leader protein
MCS - multiple cloning site
MDS - Myelodysplastisches Syndrom MOI - multiplicity of infection
MoMLV - moloney murine leukaemia virus MW - Molekulargewicht
ND - normal donor / Normalspender
NF-κB - nuclear factor 'kappa-light-chain-enhancer' of activated B-cells Nip3 - Bcl-2/adenovirus nineteen kDa interacting protein-3
NTP - N-terminales Peptid OD - optische Dichte
PARL - presenilin- associated, rhomboid-like PBS - phosphate buffered saline
PCR - polymerase chain reaction
PEST-Sequenz - Prolin (P), Glutaminsäure (E), Serin (S) und Threonin (T) PFA - Paraformaldehyd
PFU - plaque forming unit PHB2 - Prohibitin 2
PIC - Proteaseinhibitor Cocktail PKD2 - polycystic kidney disease 2 PLN - Phospholamban
Poly (I:C) - polyinosinic-polycytidylic acid PVDF - Polyvinylidenfluorid
RT - Raumtemperatur
SCN - severe congenital neutropenia SDS - Natriumdodecylsulfat
SDS-PAGE - Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese Smac - second mitochondria derived activator of Caspases
SSC – side scatter TBE - Tris-Borat-EDTA TBS - tris buffered saline
TEMED - Tetramethylethylendiamin
TICAM-1 - Toll-IL-1 receptor (TIR) domain containing adaptor molecule-1 TLR3 – toll-like receptor 3
TNF - Tumor Nekrose Faktor
TRADD - TNF receptor associated death domain TRAIL - TNF-related apoptosis inducing ligand TRITC - Tetramethylrhodamine Isothiocyanat Vol - Volumen
Vpr - human immunodeficiency virus type 1 viral protein R VSV-G - vesicular stomatitis virus G
WASP - Wiskott-Aldrich Syndrom Protein
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