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«Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von ...»

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Conditional inactivation of the ubiquitin

protein ligase Itch of the mouse

Inaugural-Dissertation

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Universität zu Köln

vorgelegt von

Kristina Jönsson

aus Svedala, Schweden

September 2002

Berichterstatter: Prof. Dr. Alexander Tarakhovsky

Prof. Dr. Thomas Langer

Tag der müntlichen Prüfung: 25 November 2002

To my parents Annagreta och Börge,

whose love and support made this work possible

A. INTRODUCTION

A.1. REGULATION OF PROTEIN LEVEL BY DEGRADATION

A.2. THE UBIQUITIN-PROTEASOME DEGRADATION PATHWAY

A.3. UBIQUITIN PROTEIN LIGASES (E3)

A.4. THE ITCH GENE AND PROTEIN

A.5. POTENTIAL SUBSTRATES OF ITCH

A.6. THE IMMUNE RESPONSE

A.7. ACTIVATION OF TH CELLS IN THE IMMUNE RESPONSE

A.8. TH1 VERSUS TH2 RESPONSE

A.9. AUTOIMMUNITY AND SYSTEMIC INFLAMMATION

A.10. CONDITIONAL GENE INACTIVATION

A.11. AIMS OF THIS STUDY

B. MATERIAL AND METHODS

B.1. BACTERIAL TRANSFORMATION

B.2. ISOLATION OF PLASMID/BAC DNA

B.3. CLONING OF GENOMIC DNA

B.4. RESTRICTION ENZYME ANALYSIS

B.6. SYNTHETIC OLIGONUCLEOTIDES

B.7. PURIFICATION OF DNA FRAGMENTS

B.8. DNA SEQUENCING

B.9. ISOLATION OF GENOMIC DNA

B.10. SOUTHERN BLOT ANALYSIS

B.11. AMPLIFICATION AND PURIFICATION OF PROBES USED FOR SOUTHERN BLOT ANALYSIS

B.12. RADIOACTIVE LABELING OF PROBES

B.13. POLYMERASE CHAIN REACTION (PCR)

B.14. ISOLATION OF RNA

B.15. CDNA SYNTHESIS

B.16. REVERSE TRANSCRIPT-PCR USING THE LIGHT CYCLER

B.17. AMPLIFICATION OF ITCH CDNA FOR CLONING OF THE ITCH EXPRESSION VECTOR

B.18. EXPRESSION AND PURIFICATION OF RECOMBINANT GST- ITCH FUSION PROTEIN

B.19. WESTERN BLOT ANALYSIS

B.20. IMMUNOHISTOCHEMISTRY

B.21. ELISA

B.21.1. Isotype ELISA

B.21.2. Anti-DNA ELISA

B.21.3. IFNg ELISA for serum

B.21.4. IFNg and IL-4 ELISA for cell culture supernatant

B.22. GENERATION OF MUTANT MICE

B.22.1. Embyonic Stem Cell Culture

B.22.2. Transfection of Embyonic Stem Cells

B.22.3. Generation of mutant mice

B. 23. GENERATION OF ITCH POLYCLONAL ANTIBODIES

B.24. PREPARATION OF SINGLE CELL SUSPENSIONS FROM MOUSE ORGANS

B.25. CYTOFLUOROMETRIC ANALYSIS AND CELL SORTING

B.26. INTRACELLULAR STAINING OF CYTOKINES

B.27. PURIFICATION OF LYMPHOCYTE SUBPOPULATIONS USING MAGNETIC CELL SORTING (MACS)

B.28. TH1 VERSUS TH2 POLARIZATION IN VITRO

C. RESULTS

C.1. ANALYSIS OF ITCH MRNA EXPRESSION IN LYMPHOCYTE POPULATIONS

C.2. GENERATION OF MICE HARBORING A LOXP-FLANKED ITCH GENE

C.2.1. Polymorphism in the itchy locus

C.2.2. Sub-cloning and sequencing of the itchy genomic locus

C.2.3. Construction of the itch targeting vector for homologous recombination in ES cells

C.2.4. Transfection of ES cells and identification of homologous recombinant clones

C.3. GENERATION OF MOUSE STRAINS WITH UBIQUITOUS OR CELL TYPE SPECIFIC INACTIVATION OF THE ITCH

GENE

C.3.1. Generation of mice with ubiquitous deletion of itch

C.3.2. Cell type specific inactivation of the itch gene

C.4. EXPRESSION OF ITCH PROTEIN IN ITCH MUTANT MICE

C.4.1. Generation of anti-Itch polyclonal antibodies

C.4.2. Expression of Itch protein in itch mutant mice

C.5. PHENOTYPICAL ANALYSIS OF ITCH MUTANT MICE

C.5.1. Systemic inflammation in itch mutant mice

C.5.2. Elevated levels of blood eosinophils and basophils and signs of anemia in itch fl/fl CD4 Cre+/- mice

C.5.3 Cell numbers in lymphoid organs of itch mutants

C.5.4. No impairment of the T cell development or B and T cell ratio in itch fl/fl CD4 Cre+/- mice................ 59 C.5.5. Changed ratios of thymocyte subsets in old itch-CD4Cre mutants

C.5.6. Serum titers of different immunoglobulin isotypes in mice with T-cell specific and ubiquitous deletion of itch

C.5.7. In vitro Th1-Th2 differentiation of itch deleted naïve CD4 single positive peripheral T cells.............. 67 D. DISCUSSION

D.1. THE ROLE OF ITCH IN THE DEVELOPMENT OF SYSTEMIC INFLAMMATORY DISEASE OF A18H MICE

D.2. EXPRESSION OF ITCH IS INCREASED IN MATURE B CELLS AND T CELLS

D.3. POLYMORPHISM IN THE ITCHY LOCUS

D.4. GENERATION OF MOUSE STRAINS WITH UBIQUITOUS OR CELL TYPE SPECIFIC INACTIVATION OF THE ITCH

GENE

D.5. THE T CELLS, BUT NOT B CELLS, IN ITCH MUTANT MICE ARE RESPONSIBLE FOR THE SYSTEMIC

INFLAMMATORY DISEASE

D.6. NO DOMINANT NEGATIVE EFFECT OF THE MUTATED ITCH PROTEIN

D.7 INACTIVATION OF THE ITCH GENE IN T CELLS INDUCES INFLAMMATION IN MICE

D.8. SPLENOMEGALY AND LYMPHODENOPATHY IN ITCH FL/FL CD4 CRE+/- MICE IS DUE TO AN INCREASE OF

LYMPHOCYTES AS WELL AS OTHER CELL TYPES

D.9. MUTANT ITCH MICE HAVE INCREASED LEVELS OF SERUM IMMUNOGLOBULIN IGM, IGG1, IGA AND IGE... 75

D.10. DISRUPTED SPLENIC ARCHITECTURE IN ITCH FL/FL CD4 CRE MICE

D.11. ITCH IS NOT REQUIRED FOR NORMAL LYMPHOCYTE DEVELOPMENT





D.12. ALTERATIONS OF CELL POPULATIONS IN THE THYMUS AND ACTIVATED PHENOTYPE OF T CELLS.............. 76 D.13. TH2 VERSUS TH1 BIASED IMMUNE RESPONSE IN ITCH FL/FL CD4CRE MICE

D.14. OLD ITCH FL/FL CD4 CRE MICE SHOW SIGNS OF ANEMIA

D.15. THE PHENOTYPE OF ITCH FL/FL CD4CRE MICE RESEMBLES THAT OF A18H MICE ON C57BL/6 GENETIC BACKGROUND

D.16. THE POSSIBLE ROLE OF INFECTIOUS AGENTS OR PATHOGENS IN THE INDUCTION OF DISEASE

D.17. POTENTIAL MECHANISM(S) UNDERLYING THE DEVELOPMENT OF THE SYSTEMIC INFLAMMATION DISEASE

FL/FL CD4CRE MICE

IN ITCH

E. ABSTRACT

F. KURZE ZUSAMMENFASSUNG

G. SUMMARY

I. REFERENCES

J. ACKNOWLEDGEMENT

L. ERKLÄRUNG

K. LEBENSLAUF

A. Introduction Incorrect activation or lack of regulation of signaling cascades in the immune system can induce severe immune disease, such as autoimmunity and inflammatory diseases (for review see (Strober et al., 2002). It has for example, been observed that mice lacking PD-1, an immunoinhibitory receptor, develop autoimmune disorders characterized by production of high titers of autoantibodies (Freeman et al., 2000). In addition, transgenic mice expressing the T cell costimmulatory molecule CD40L, develop a chronic inflammatory disease (Clegg et al., 1997).

Signaling pathways are complex networks of proteins, where biological functions can be regulated by different mechanisms, examples are activation and deactivation of proteins by phoshorylation/dephosphorylation, restriction of protein localization to promote or inhibit protein interactions as well as complete abolishment of activated proteins by degradation. A good example of this is signaling regulation of the transcription factor nuclear factor k B (NFkB), which involves all these three mechanisms. NFkB is inactive when bound to the inhibitors of NFkB (IkB) in the cytosol. Phosphorylation of IkB leads to its ubiquitination and degradation in the proteasome, thereby releasing NFkB which can be translocated to the nucleus where it can act in transcription (Chen et al., 1995; DiDonato et al., 1996; Li et al., 1995).

Duration of signaling pathways are normally tightly controlled by a negative feedback loop.

In its simplest form a signal induces its own negative regulator leading to a decrease of signaling after the signal has reached a threshold. An example of this is the control of cytokine signaling through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, where the intracellular protein tyrosine kinases, JAKs, are recruited to the cytokine receptors and transduce the signal through STATs. This signaling induces the expression of suppressors of cytokine signaling-1 (SOCS-1), which inturn interacts with the receptor-bound activated JAKs, resulting in inhibition of tyrosine kinase activity (Endo et al., 1997; Naka et al., 1997; Nicholson et al., 1999). In addition, SOCS-1 has been shown to interact with the ubiquitination machinery and is thought to target associated signaling molecules to proteasomal degradation (Kamura et al., 1998; Zhang et al., 1999).

A.1. Regulation of protein level by degradation Degradation of cellular proteins is an important process to control the levels of specific proteins as well as to eliminate damaged or misfolded proteins. There are two main pathways of protein degradation in the cell, lysosomal degradation of mainly membranal proteins and degradation by proteasomes of polyubiquitinated intracellular proteins. Polyubiquitination of substrates is usually needed for recognition by the proteasome, while mono- or diubiquitinated proteins can be recognized by and directed to the endocytotic pathway reviewed in (Dunn and Hicke, 2001; Hicke, 1999; Hicke, 1997; Lemmon and Traub, 2000).

Poly-ubiquitinated substrates, which are recognized and degraded in the proteasome, include cell cycle regulators as M-phase cyclins and G1 cyclins (King et al., 1994; Won and Reed, 1996), transcription factors such as, STAT1 (Kim and Maniatis, 1996) and c-Jun (Treier et al., 1994), signal transducers such as Protein kinase C (Lee et al., 1996), and protein tyrosin kinase Src (Hakak and Martin, 1999; Harris et al., 1999) as well as abnormal proteins, which have to be removed from the cells. This pathway is also involved in the regulation of signaling cascades by degradation of inhibitory proteins as observed for the activation of the NFkB transcription factor, upon IkB-a ubiquitination and degradation (Chen et al., 1995;

DiDonato et al., 1996; Li et al., 1995).

Identified ubiquitinated substrates degraded in the lysosome are receptors or receptor subunits such as, platelet-derived Growth factor b-receptor (PDGF b-receptor), (Mori et al., 1993).

Thus, ubiquitin-mediated degradation of proteins plays an important role in regulation of signal transduction and in contrast to other mechanisms, for example phosphorylation/dephosphorylation, leads to an irreversible end of the signaling cascade.

A.2. The ubiquitin-proteasome degradation pathway Ubiquitin-dependent degradation of cellular proteins operates as a two step mechanism. The first step includes attachment of an ubiquitin molecule, a highly conserved 76-amino acid polypeptide, to a substrate. Ubiquitin can then serve as its own substrate for ubiquitin conjugation (ubiquitination), which results in the formation of polyubiquitin chains. The second step of the process includes recognition of the polyubiquitinated protein and its degradation by the 26S proteasome, or internalisation and degradation by the lysosomal pathway, as has been shown for some monoubiquitination membranal proteins, for review see (Dunn and Hicke, 2001; Hicke, 1999; Lemmon and Traub, 2000). Ubiquitination of cellular proteins is a highly regulated process (Fig.1). In this process, ubiquitin, is bound to an ubiquitin activating enzyme also called E1, in an ATP- dependent process resulting in a high energy thiol ester bond between a cysteine in the E1 and the ubiquitin molecule. In the next step of the cascade, the ubiquitin molecule is transferred to an ubiquitin conjugating enzyme also called ubiquitin carrier protein or E2, where it is covalently linked to a conserved cysteine in the E2. Finally, the ubiquitin is transferred to the substrate molecule with the help of a third class of enzymes, the ubiqitin protein ligases or E3s (Hershko and Ciechanover, 1998;

Hershko and Ciechanover, 1992; Jentsch, 1992). Ubiquitin can then serve as a substrate itself in the formation of polyubiquitin chains, possibly with the aid of an E4 (Koegl et al., 1999).

The ubiquitin system appear to be a hierarchic system with only one functional E1 known so far, several E2s (between 20-30 estimated in mammals) and a large number of E3s (more then hundered revealed by database searches).

Figure 1. The ubiquitination pathway.

Shown is the specific enzymatic reaction of ubiquitin (Ub) conjugation to a substrate molecule, which involves three types of enzymes, the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin-protein ligase (E3). The conjugation of Ub to a cysteine in the E1 is an ATP dependent processs, the Ub is then transferred to a conserved cysteine in the E2 and, finally, in interaction with an E3, the Ub is transferred to lysine residues in the substrate molecule. After polyubiquitination, the substrate is degraded in the proteasome leaving Ub and peptides as end products.

A.3. Ubiquitin protein ligases (E3) E3s have loosely been defined as proteins participating together with E1 and E2s in the ubiquitination of proteins, which cannot be recognized and ubiquitinated by E2 alone (Ciechanover, 1994; Hershko and Ciechanover, 1992). A common feature of all known E3s is that each E3 appears to specifically interact with distinct E2s or distinct subsets of E2s (Feldman et al., 1997; Huibregtse et al., 1995; Kumar et al., 1997; Lisztwan et al., 1998;

Moynihan et al., 1999; Nuber et al., 1996; Scheffner et al., 1994; Schwarz et al., 1998). The substrate recognition of the ubiquitination system is achieved by direct interaction between the substrate and the E3 or with E3 as part of a complex with an E2. The interaction between the substrate and the E3 is likely to contribute to the high substrate specificity in the system.

Based on the domain essential for E3 activity, two types of E3s have been defined, the ring finger E3s and the homology to E6-AP C-terminus (hect) domain E3s (Huibregtse et al., 1995;

Joazeiro and Weissman, 2000).

The family of ring finger domain E3s can be devided into four different groups, examples of E3s from these groups are the Rbx1, UBR1, Apc11p and c-Cbl. The Rbx1 (or Hrt1/Roc1) protein, is an essential component of the large combinatorial Skp1-cullin-F-box (SCF) E3 complexes, acting in the ubiquitination of phosphorylated proteins (Kamura et al., 1999;



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