El principio de celeridad

For the identification of dominant apoptosis-inducing factors, the direct functional genetic screening procedure was applied as described by Grimm and Leder [Grimm, 1997]. In this screen, a normalized cDNA library was prepared from murine kidney tissue and was transformed into E.coli cells. The transformed E.coli cells were diluted and distributed into 96 well plates, in a way that per well only about 1 to 5 different cDNA clones were amplified. Plasmid DNA was prepared from those small bacteria pool clones and was transfected into human embryonic kidney 293T cells which subsequently were inspected microscopically for an apoptotic phenotype. The plasmid DNA pools which upon transfection were identified to induce signs of apoptosis in 293T cells, were retransformed into E.coli and plasmid DNA from single colonies was extracted in order to identify the single cDNA clone that was responsible for apoptosis induction (for more details see Materials and Methods 2.2.6).

The large-scale application of this screen in our laboratory led to the isolation of several cDNAs which dominantly induce apoptosis upon transient transfection into 293T cells. Some of those cDNAs were identified to be genes which were already known to be involved in apoptotic pathways and have the capacity to dominantly induce apoptosis upon overexpression such as FADD, ANT-1, CIDE-A and -B or ZIP kinase and which serve as positive controls for the functionality of the screen [Bauer, 1999; Chinnaiyan, 1995; Inohara, 1998; Kawai, 1998]. The adenine-nucleotide translocator 1, ANT-1, serves as an instructive example for the specificity by which the described screening procedure allows the identification of distinct apoptosis-inducing factors of potential physiological relevance: whereas overexpression of ANT-1 results in the induction of cell death, its highly conserved homologue ANT- 2 does not induce apoptosis. This observation indicates that the observed cell death is not unspecifically induced by an inappropriate accumulation of ANT-1 and concurrent misfolding of the ANT-1 protein within the inner mitochondrial membrane, because – if this were true – the same effect would have to be expected in case of the almost identical ANT-2 protein. Thus, ANT-1 appears to specifically exert its proapoptotic activity by acting as a component of the permeability transition pore complex which is known to play an essential role in the mediation of apoptosis signals at the mitochondria [Bauer, 1999].

One of the cDNA clones that was selected from the genetic screen (by myself in collaboration with Ulla Cramer) was designated UI64, which not only induced the typical apoptotic morphological changes in 293T cells but also produced oligonucleosomal DNA fragmentation as a hallmark event of apoptosis (Fig 3.1.1).

DNA sequencing of the UI64 cDNA clone and sequence analysis (Fig 3.1.2) using BLAST [Altschul, 1997] identified UI64 as the mouse orthologue (gi:7949157) of the ubiquitin-specific protease UBP41 (also denominated USP2) which recently had been isolated and characterized in chicken skeletal muscle [Baek, 1997].

Fig. 3.1.1

The cDNA clone UI64 causes cell death when transiently transfected into 293T cells.

(A) 293T cells were transfected with a control plasmid (left)or with the conpicuous plasmid clone UI64 from the screen for dominant apoptosis inducing genes (right). 48h post transfection, the phenotype was observed by phase contrast microscopy. (B) 293T cells were transfected with a control plasmid or with UI64. 48h post transfection the cells were harvested, DNA was extracted and separated on a 2% agarose gel, stained with ethidiumbromide and visualized on a transilluminator.

Cys-Domain

UI64 ---MNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQR

mUBP41 ----MLNKAKNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQR

hUBP41 ----MLNKAKNSKSAQGLAGLRNLGNTCFMNSILQCLSNTRELRDYCLQR

gUBP41 MAARMAPTPRSSKVVQGLTGLRNLGNTCFMNSILQCLSNTKELRDYCLQN

UI64 LYMRDLGHTSSAHTALMEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAP

mUBP41 LYMRDLGHTSSAHTALMEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAP

hUBP41 LYMRDLHHGSNAHTALVEEFAKLIQTIWTSSPNDVVSPSEFKTQIQRYAP

gUBP41 QYLRDLNNNSRMRTALMSEFAKLIQLLWTSSPNDSVSPSEFKTQIQRYAP

Asp-Domain

UI64 RFMGYNQQDAQEFLRFLLDGLHNEVNRVAARPKASPETLDHLPDEEKGRQ

mUBP41 RFMGYNQQDAQEFLRFLLDGLHNEVNRVAARPKASPETLDHLPDEEKGRQ

hUBP41 RFVGYNQQDAQEFLRFLLDGLHNEVNRVTLRPKSNPENLDHLPDDEKGRQ

gUBP41 RFVGYNQQDAQEFLRFLLDGLHGEVNRVLVRPRANADTLDHLPDDEKSRQ

Homology-Domain I

UI64 MWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDXSLPIAKR

mUBP41 MWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKR

hUBP41 MWRKYLEREDSRIGDLFVGQLKSSLTCTDCGYCSTVFDPFWDLSLPIAKR

gUBP41 MWRRYQEREDSRVSDLFVGQLKSSLTCSECGYCSTAFDPFWDLSLPIPKK

Homology-

UI64 GYPEVTLMDCMRLFTKXDILDGDEKPTCCRCRARKRCIKKFSVQRFPKIL

mUBP41 GYPEVTLMDCMRLFTKEDILDGDEKPTCCRCRARKRCIKKFSVQRFPKIL

hUBP41 GYPEVTLMDCMRLFTKEDVLDGDEKPTCCRCRGRKRCIKKFSIQRFPKIL

gUBP41 GYGEVTLMDCLRLFTKEDVLDGDEKPTCCRCKARTRCTKKFSIQKFPKIL

Domain II His-

UI64 VLHLKRFSESRIRTSKLXTFVNFXLRDLDLREFASENXNHAVYNLYAVSN

mUBP41 VLHLKRFSESRIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSN

hUBP41 VLHLKRFSESRIRTSKLTTFVNFPLRDLDLREFASENTNHAVYNLYAVSN

gUBP41 VLHLKRFSEARIRASKLTTFVNFPLKDLDLREFASQSCNHAVYNLYAVSN

Domain Homology-Domain III

UI64 HSGTXMGGXYTAYCRSPVTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYEL

mUBP41 HSGTTMGGHYTAYCRSPVTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYEL

hUBP41 HSGTTMGGHYTAYCRSPGTGEWHTFNDSSVTPMSSSQVRTSDAYLLFYEL

gUBP41 HSGTTMGGHYTAYCKSPISSEWHSFNDSRVTPMSSSHVRSSDAYLLFYEL

UI64 ASPPSRM

mUBP41 ASPPSRM

hUBP41 ASPPSRM

gUBP41 ASPSSRM

For the isolation of the human orthologue of UBP41, PCR primers were designed based on the Genbank entry for hUBP41 (gi: 4759291) with the forward primer containing the start ATG and the reverse primer representing a region downstream of the stop codon. The obtained PCR product confirmed the published sequence with the exception of a few residues at the extreme carboxy- terminus which was determined to be identical to the mouse amino acid sequence. Therefore, the PCR product was regarded to represent human UBP41, subsequently designated hUBP41. A carboxy- terminal HA-tag sequence was added to hUBP41, resulting in the HA-fusion protein hUBP41-HA for convenient detection in immunoblot analysis using anti-HA antibodies. Interestingly, the alignment of its human, murine, and chicken amino acid sequences (Fig. 3.1.2) indicates that UBP41 is highly conserved not only within the catalytic or homology domains but also in the regions in between those domains.

Fig. 3.1.2

The cDNA clone UI64 is identified as the mouse orthologue of UBP41.

BLAST sequence analysis revealed that the isolated apoptosis-inducing clone UI64 encodes the mouse orthologue of the ubiquitin- specific protease UBP41. The UBP41 protein sequence is highly conserved between homo sapiens (hUBP41), mus musculus (mUBP41), and gallus gallus (gUBP41). Identical residues are marked by blocks. Homology domains characteristic for ubiquitin-specific proteases are indicated, such as the cysteine, aspartate and histidine domain, as well as three additional homology domains. The catalytic cysteine is marked by an asteriks.