As a PostDoc in the group of Dr. habil. Jürgen Ruland I am involved in various projects that investigate genes involved in the formation and progression of lymphoma due to chromosomal translocations or other genetic aberration. Our aim is to define the physiological and pathological role of those genes either by knocking the genes out in mice or by generating transgenic mice that overexpress the gene. The main focus of our research is the NFkB signalling pathway induced by receptors of the immune system (such as the antigen receptor, the T cell receptor, innate pattern recognition receptors). Indeed, many genes investigated in our lab are essential signalling components in immune cells, e.g. Bcl10, MALT1, and Card9 with more genes currently under investigation. My main project is the establishment of a conditional Cre-Lox based knockout of a gene that has been identified to be translocated in lymphoma patients. We investigate its role in lymphocyte development and differentiation and in addition found in the total knockout, that the gene is critically involved in the embryonic development of the mouse.
Methods used:
Gene targeting, embryonic stem cell culture, mouse work, bone marrow transplantation, flow cytometry,
in vitro cell signalling, immunofluorescence, reporter assays, retroviral infections,
cloning, protein purification, kinase assays, immunopreciptitation, general genetic and molecular biology
techniques.
Publications thus far during the PostDoc time:
Gross O, Gewies A, Finger K, Schafer M, Sparwasser T, Peschel C, Forster I, Ruland J
Nature. 2006 Aug 10;442(7103):651-6
Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.
Fungal infections are increasing worldwide due to the marked rise in immunodeficiencies including AIDS; however, immune responses to fungi are poorly understood\. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 represents the prototype of innate non-Toll-like receptors (TLRs) containing immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive antigen receptors. Here we identify Card9 as a key transducer of Dectin-1 signalling. Although being dispensable for TLR/MyD88-induced responses, Card9 controls Dectin-1-mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. Card9 couples to Bcl10 and regulates Bcl10-Malt1-mediated NF-kappaB activation induced by zymosan. Yet, Card9 is dispensable for antigen receptor signalling that uses Carma1 as a link to Bcl10-Malt1. Thus, our results define a novel innate immune pathway and indicate that evolutionarily distinct ITAM receptors in innate and adaptive immune cells use diverse adaptor proteins to engage selectively the conserved Bcl10-Malt1 module.
Gewies A, Ruland J
AfCS-Nature Molecule Pages, 2005, doi:10.1038/mp.a000366.01
Bcl10
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As part of the junior research group of Dr. Stefan Grimm I was working on my Ph.D. thesis
which has been titled: "Investigation of the ubiquitin-specific protease UBP41 and of
the lysosomal cysteine proteases cathepsin-L and cathepsin-B
as potential mediators of proapoptotic signalling".
Apoptosis is a form of programmed cell death by which cells
of a multicellular organism die in a well-ordered and genetically determined sequence of morphologic
and molecular events. Since apoptosis plays major roles during development and homeostasis, it is
highly regulated by the action of a multitude of proteins that either promote or suppress the activation
of the cell death program. Although a plethora of pro- or antiapoptotic proteins have already been
described, many more, yet unidentified protein factors are expected to be involved in the induction,
mediation and regulation of apoptosis. In this context, the main objective of my thesis was to identify
and characterize proteins as novel regulatory factors of apoptosis pathways. For this, a genetic
screen was applied that aimed on the isolation and identification of cDNA clones which upon
overexpression induce the typical morphological features of apoptosis in the chosen 293T cell system.
Selected cDNA clones identified in this way, were subsequently characterized for their proapoptotic
activity.
Within my thesis I worked on two genes identified by the screening procedure: the first project characterized the
ubiquitin-specific protease UBP41 as a protein which upon overexpression causes apoptosis induction
in several mammalian cancer cell lines. The second project investigated a possible involvement of the
lysosomal cysteine proteases cathepsin-L and cathepsin-B in apoptosis pathways induced by distinct
death stimuli, in particular by the tumor necrosis factor ? (TNF-?). Therefore, both projects examine a
possible regulation of apoptosis induction by proteases that are part of one of the two major systems of
protein degradation. UBP41 as a protease with deubiquitylating activity is expected to play a role in
the ubiquitin/proteasome system which is the major proteolytic apparatus for the degradation of
cytosolic proteins. Cathepsins, on the other hand, are lysosomal proteases that participate in the
breakdown of membrane-associated proteins and of extracellular proteins that are taken up by
endocytosis. Both, the ubiquitin/proteasome system as well as lysosomal proteases have been
previously implicated in the regulation and mediation of apoptosis, and it therefore appeared
particularly attractive to further study the effect of UBP41 and cathepsins on cell death signalling in
more detail.
Methods used:
Cell culture techniques, genetic high throuput screening, diverse apoptosis detection assays (DNA laddering, radioactive DNA fragmentation test, DAPI staining ...), proliferation tests, flow cytometry, immunofluorescence, diverse standard methods
Publications resulting from the Ph.D. thesis work:
Gewies A, b.. Grimm S
Br J Cancer. 2003 Oct 20;89(8):1574-80
Cathepsin-B and cathepsin-L expression levels do not correlate with sensitivity of tumour cells to
TNF-alpha-mediated apoptosis.
Recently, evidence has been accumulated that besides the caspase proteases, lysosomal cathepsins may play a role in apoptosis induction. This is especially significant as many human tumour cells express high levels of cathepsins, which might sensitise these cells to specific proapoptotic stimuli mediated by cathepsins. We found that TNF-alpha-mediated DNA fragmentation in tumour cells was significantly reduced in the presence of E64d and CA074Me, two inhibitors of lysosomal cysteine proteases. Transient transfection of cathepsin-B (Cath-B) and -L (Cath-L) resulting in expression levels comparable to those found in many tumours did not sensitise tumour cells to TNF-alpha-mediated apoptosis. As lysosomal proteases are thought to be activated by their release from this organelle into the cytosol, we used the lysosomotropic detergent N-dodecyl-imidazole-HCl (NDI-HCl) to disturb lysosomal integrity efficiently and trigger the release of its proteolytic content into the cytosol. Treatment of HeLa cells with NDI-HCl resulted in cell death, which, however, could also not be influenced by augmented Cath-B or -L expression level. Therefore, our data do not support the hypothesis that the high Cath-B or -L expression levels frequently detected in tumour cells might be exploited to target selectively those tumours for an enhanced cell death effect induced by lysosomotropic agents.
Mund T, Gewies A, Schoenfeld N, Bauer MK, Grimm S
FASEB J. 2003 Apr;17(6):696-8
Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum.
We have isolated Spike, a novel and evolutionary conserved BH3-only protein. BH3-only proteins constitute a family of apoptosis inducers that mediate proapoptotic signals. In contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Furthermore, unlike the known BH3-only proteins, Spike could not interact with all tested Bcl-2 family members, despite its BH3 domain being necessary for cell killing. Our findings indicate that Spike is localized to the endoplasmic reticulum. The endoplasmic reticulum is an organelle that has only recently been implicated in regulation of apoptosis. At this locale, Spike interacts with Bap31, an adaptor protein for pro-caspase-8 and Bcl-XL. In doing so, Spike is able to inhibit the formation of a complex between Bap31 and the antiapoptotic Bcl-XL protein. Furthermore, Spike transmits the signal of specific death receptors. Its down-regulation in certain tumors suggests that Spike may also play a role in tumorigenesis. Our findings add new insight for how BH3-only and antiapoptotic Bcl-2 proteins regulate cell death.
Gewies A, Grimm S
Cancer Res. 2003 Feb 1;63(3):682-8
UBP41 is a proapoptotic ubiquitin-specific protease.
We have isolated Ubp41, a ubiquitin-specific protease, in a screen for proapoptoticgenes. We found that overexpression of Ubp41 is sufficient to elicit all features of apoptosis in human cells. In contrast, an enzymatically defective UBP41 mutant and homologous ubiquitin-processing protease family members did not significantly induce cell death. Overexpression of Ubp41 resulted in a strong deubiquitination of a broad range of proteins, but surprisingly did not lead to a stabilization of protein substrates known to be regulated by the ubiquitin-proteasome system such as the cell cycle factors p21 and p27. Hence, in contrast to the proteasome inhibitor MG132, Ubp41 overexpression did not arrest cells in G(2)/M\. Rather, overexpression of hUbp41 seems to interfere with the ubiquitin-system and to cause the activation of apoptosis pathways by stabilizing specific substrates. Hence, for the first time we found that a member of the deubiquitinating enzymes has a direct proapoptotic activity additionally tightening the connection between apoptosis and the ubiquitin-proteasome system.
In the research group of Michael B. Cohen MD (Laboratory of Experimantal Biology of Prostate Cancer) I was involved in the characterization of apoptotic pathways in the prostatic carcinoma cell lines PC3, DU145, JCA1 and ALVA31. Using those cell-lines, I was examining Fas-mediated apoptosis in situ as well as in a cell-free system using cytoplasmic extracts and isolated nuclei. In situ it could be shown that BID cleavage and cytochrome c released from the mitochondria are early events during Fas-induced cell death. The results from the cell-free system demonstrate that the signalling pathways induced by cytochrome c (caspase cascade, nuclear apoptotic events)are intact in the examined cell-lines. Together, the data suggest that the mitochondrial apoptosis pathway is involved in Fas-mediated cell death of prostatic cancer cell lines.
Another goal was to determine whether the ceramide second messenger pathway participates in the Fas-mediated apoptosis in prostate cancer cell lines. The synthetic ceramide analogue C2-ceramide induced cell-death in the cell-lines PC3 and DU145 resulting also in DNA fragmentation. DNA fragmentation could be blocked by the caspase-inhibitor zVAD-fmk but cell-death was not prevented by zVAD-fmk! It can be assumed that in this cell system C2-ceramide induces cell-death involving apoptotic and non-apoptotic mechanisms. Moreover, no increase in endogeneous ceramide levels could be observed after treatment of PC3 cells with agonistic anti-Fas antibodies. The data provide evidence against an involvement of ceramide in Fas-mediated apoptosis of PC3 cells.
Besides that I started studies on NF-kB activation upon the induction of apoptosis in prostate caner cell lines by using EMSA.
Methods used:
Cell culture techniques, diverse apoptosis detection assays (DNA laddering, radioactive DNA fragmentation test, DAPI staining ...), proliferation tests, isolation of nuclei for the cell free system, preparation of biologically active cell extracts, western blotting, Caspase enzyme assay, EMSA, lipid analysis.
Publications during the time in Iowa City:
Gewies A, Rokhlin OW, Cohen MB
Lab Invest. 2000 May;80(5):671-6
Ceramide induces cell death in the human prostatic carcinoma cell lines PC3 and DU145 but does
not seem to be involved in Fas-mediated apoptosis.
Treatment of cells with synthetic C2-ceramide has been reported to induce apoptosis in several cell systems, and endogenously formed ceramide has been proposed to act as a second messenger, activating signaling pathways which contribute to the execution of apoptotic cell death after Fas ligation or tumor necrosis factor receptor-1 ligation. In this study, we examined the effect of exogenously administered C2-ceramide on the human prostatic carcinoma cell lines PC3 (Fas-sensitive) and DU145 (Fas-resistant). In both cell lines, C2-ceramide induced cell death in a dose-dependent manner, whereas a structural analog, C2-dihydroceramide, did not. The pan-caspase inhibitor zVAD-fmk did not prevent C2-ceramide-induced cell death but did prevent C2-ceramide-induced DNA fragmentation, indicating that apoptotic and non-apoptotic mechanisms are involved in C2-ceramide-induced death. Interestingly, cycloheximide prevented C2-ceramide-induced DNA fragmentation, indicating that ceramide-induced apoptosis in PC3 and DU145 requires new protein synthesis. In addition, because cycloheximide converts Fas-resistant DU145 to Fas-sensitive as assessed by DNA fragmentation, ceramide does not seem to play a major role in the Fas-mediated pathway in this cell line. We also determined the levels of endogenous sphingomyelin after Fas ligation in PC3. No decrease of sphingomyelin levels could be detected after Fas activation\. We conclude that sphingomyelinase-generated ceramide does not play a role in Fas-mediated apoptosis in PC3, and that there are fundamental differences in the mechanisms of cell death induced by C2-ceramide and Fas ligation.
Gewies A, Rokhlin OW, Cohen MB
Cancer Res. 2000 Apr 15;60(8):2163-8
Cytochrome c is involved in Fas-mediated apoptosis of prostatic carcinoma cell lines.
We have shown previously that the pathways leading to Fas-mediated apoptosis in prostatic carcinoma cell lines are intact, because apoptosis can be triggered either by Fas ligation alone in the Fas-sensitive cell lines PC3 and ALVA31 or by rendering the Fas-resistant cell lines DU145 and JCA1 Fas-sensitive by combined treatment with anti-Fas monoclonal antibody and cycloheximide (O.W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997). In this study, we demonstrate that two of the early events after Fas ligation are the release of cytochrome c from the mitochondria and activation of caspase-9. We also found that Bid is processed after Fas ligation and thus might activate the mitochondria-dependent apoptotic cascade. In a cell-free system, cytochrome c induced caspase-3-like activity in cytoplasmic extracts from all four cell lines studied, although differences in the level of enzymatic activity were observed. Western blot analysis revealed that caspase-7 is activated by cytochrome c at the same level in all extracts, whereas expression and activation of caspase-3 varied considerably. Cytochrome c-activated extracts displayed different abilities in the induction of apoptotic features in isolated nuclei such as morphological changes and DNA fragmentation. However, differences in nuclear apoptotic activity induced by cytochrome c did not correlate with the level of caspase-3 like activity in the different extracts. These results suggest that the mitochondrial pathway is involved in Fas-mediated apoptosis in prostatic carcinoma cell lines and that, in addition to caspase-7 and caspase-3, there are other factors that confer nuclear apoptotic activity.
Rokhlin OW, Gudkov AV, Kwek S, Glover RA, Gewies AS, Cohen MB
Oncogene. 2000 Apr 6;19(15):1959-68
p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic
carcinoma cell line LNCaP.
The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.
In the Department of Molecular Genetics I first was doing community service, later on I was Research Assistant. I was responsible for the documentation of incoming samples (e.g. blood, tissues) and for the preparation of genomic DNA from blood but also other sources (e.g. fibroblasts, bone). Besides that I had to do administrative and basic lab services and participated in the instruction of students.
I was involved in different projects, especially in a project for the screening of mutations in the Neurofibromatosis 1 (NF1) gene. Its aim was to screen the whole NF-1 gene among a large number of clinically well characterized NF1 patients in order to find possible hotspot regions, to find a possible genotype-phenotype correlation, and to allow a functional analysis of the NF1 gene or its product neurofibromin. Besides this, the study was supposed to lead to the establishment of a reliable and cost-effective detection system for NF1 pointmutations or smaller deletions/insertions that could be used in routine diagnostics. The main screening method used was Temperature-Gradient Gel Electrophoresis (TGGE). Using TGGE and subsequent DNA sequencing, I contributed to the detection of several pointmutations or deletions/insertions. I also started to establish a diagnostic routine.
Additionally, I detected a mutation in the gene of the Gs-alpha subunit in fibrous dysplastic bone material from a patient with McCune-Albright syndrome. The mutation was only present in a mosaic pattern, and by slot-blot hybridization the level of mutated cells in different tissues was examined. Interestingly, this McCune-Albright patient was extreme tall (213 cm), and here it was shown for the first time - clinically and genetically - that the well characterized Gs-alpha mutation in the McCune-Albright syndrome not only gives rise to a phenotype with dwarfism or normal growth (as reported before), but can also lead to adult gigantism.
Methods used:
Preparation of genomic DNA from blood, cells, tissues and bone, optimization of PCRs, DNA sequencing, restriction analysis, TGGE, slot-blot-hybridization.
Publications during the time in the Human Genetics Department:
Toliat MR, Erdogan F, Gewies A, Fahsold R, Buske A, Tinschert S, Nurnberg P.
Electrophoresis. 2000 Feb;21(3):541-4
Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence
of mutations in exon 4b.
A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.
Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kucukceylan N, Abdel-Nour M, Gewies A,
Peters H, Kaufmann D, Buske A, Tinschert S, Nurnberg P
Am J Hum Genet. 2000 Mar;66(3):790-818
Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but
points to a functional domain upstream of the GAP-related domain.
More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered n ovel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.
Buske A, Gewies A, Lehmann R, Ruther K, Algermissen B, Nurnberg P, Tinschert S
Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1).
We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1.
Tinschert S, Gerl H, Gewies A, Jung HP, Nurnberg P
Am J Med Genet. 1999 Mar 12;83(2):100-8
McCune-Albright syndrome: clinical and molecular evidence of mosaicism in an unusual giant patient.
Molecular genetics recently uncovered the mystery of the protean picture of McCune-Albright syndrome by identification of the somatic gain of function mutations in the GNAS1 gene. Here we present an adult patient with fibrous dysplasia and an endocrinopathy resulting in unusual giant height. The clinical diagnosis in the patient could be confirmed by molecular investigations in tissues involved in the process of fibrous dysplasia.
Klose A, Ahmadian MR, Schuelke M, Scheffzek K, Hoffmeyer S, Gewies A, Schmitz F, Kaufmann D, Peters H, Wittinghofer A, Nurnberg P
Hum Mol Genet. 1998 Aug;7(8):1261-8
Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1.
Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, cafe-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene\. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras.GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.
Klose A, Robinson N, Gewies A, Kluwe L, Kaufmann D, Buske A, Tinschert S, Peters H
Hum Genet. 1998 Mar;102(3):367-71
Two novel mutations in exons 19a and 20 and a BsaBI polymorphism in a newly
characterized intron of the neurofibromatosis type 1 gene.
Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. A total of 170 unrelated NF1 patients were screened for mutations in four exons by temperature-gradient gel electrophoresis. Preparatory work revealed the presence of a previously uncharacterized intron (19a) in what was previously designated exon 19; this allowed us to develop assays for genomic mutation screening in the newly defined exons 19a and 19b. Two novel NF1 mutations were detected: a single-base insertion in exon 19a creating a frameshift, and a second mutation affecting the splice donor site of intron 20 and leading to skipping of exon 20. A novel BsaBI polymorphism was identified in intron 19a.
During my research in Priv. Doz.Dr. Ullrich Keller's Laboratory I was mainly working on the homologue overexpression of immunophilins in Streptomyces which belong the higher prokaryotic family of Actinomycetes. Additionally I worked on a system to disrupt Streptomyces immunophilin genes.
Steptomyces chrysomallus four immunophilin genes had been identified: Cyclophilin A (ScCypA), Cyclophilin B (ScCypB), ScFKBP12, and ScFKBP33. Cyclophilins are responsible for the immunosuppressive effect of Cyclosporin A (CsA) and its homologs, whereas FKBPs mediate the immunosuppressive effect of FK506 and Rapamycin and their homologs. The biological, cellular function(s) of immunophilins was obscure for a long time, but since immunophilins were found highly conserved in all different kinds of organisms - mammals, plants, nematodes, protista, yeast and prokaryotes - they are supposed to have important functions. Since all immunophilins possess Peptidyl Prolyl cis/trans Isomerase (PPIase) activity their most likely biological role was expected to be in the folding and transport of proteins. Indeed, immunophilins were found to be involved in protein folding (e.g. NinaA in the ER of Drosophila), in the regulation of ion channels (e.g. CypD is associated with the mitochondrial permeability transition pore) but also in other special processes (e.g. cyclophilins were reported to possess DNase activity or as essential cofactors in the replication of the HIV genome).
During my Diplom project I overexpressed all four Streptomyces chrysomallus immunophilin genes in Streptomyces lividans. The objective was to possibly observe changes in the phenotype, in the metabolism or in the complex life cycle of the overexpressing Streptomyces strains. No remarkable phenotypic changes could be observed in strains overexpressing the different immunophilins. Of special interest was the overexpression of ScCypB which in contrast to the highly expressed ScCypA had previously not been shown to be be expressed on the protein level. The overexpression of ScCypB helped to demonstrate the low level expression of CypB in wildtype S. chrysomallus. Therefore, it was demonstrated that two cytosolic cyclophilins - ScCypA and ScCypB - both are expressed at the same time. In contrast, multiple different cyclophilins expressed in other organisms were reported to be localized in separate cellular compartments. This result suggests specialized roles of ScCypA and ScCypB in Streptomyces possibly by interacting with different specific ligands within the cell. This hypothesis of different ligand specifity of the two cyclophilins is supported by the differences found in their primary structure: the direct comparison between ScCypA and ScCypB shows only 45% identity, and a phylogenetic analysis placed ScCypA in the cluster of eukaryotic cyclophilins while ScCypB did neither fit to the eukaryotic nor prokaryotic cluster. Indication for the existence of ScCypA ligands in S. chrysomallus was obtained by examining its cytosolic fractions separated on a DEAE column: by western blot ScCypA was detected in high quantities in discrete fractions which did not exhibit PPIase activity. A possible explanation is the inhibition of ScCypA's PPIase activity by ligands bound to ScCypA. For the identification of ScCypA ligands, immunoprecipitation experiments were performed by incubating the fractions of interest with anti-ScCypA polyclonal antibodies. These immunoprecipitation studies were only preliminary and could not be completed within this work.
Additionally to these expression studies, a vector system for the disruption of immunophilin genes in Streptomyces was constructed: internal fragments of the four immunophilin genes were cloned into a temperature-sensitive suicide vector and transformed into S. lividans. This system was further used to produce and select for immunophilin disruption mutants.
Methods used:
Cell culture techniques (E. coli, Streptomyces), plasmid extraction, restriction, ligation, transformation (calciumchloride method and protoplast method), PCR, DNA Sequencing, preparation of cell-extracts, protein quantification, western blot, DEAE cellulose chromatography, immunoprecipitation, PPIase enzyme assay.
Publications during the time of the Diplomarbeit:
Pahl A, Gewies A, Keller U.
Microbiology. 1997 Jan;143 ( Pt 1):117-26
ScCypB is a novel second cytosolic cyclophilin from Streptomyces chrysomallus which is
phylogenetically distant from ScCypA.
A novel second streptomycete cyclophilin gene-designated sccypB-was isolated from a cosmid gene library of Streptomyces chrysomallus by using as gene probe a fragment of the previously isolated cyclophilin gene sccypA of the same organism\. From its sequence the gene sccypB should encode a protein of M(r) 18868\. Expression of sccypB in Escherichia coli as a hexaHis-tagged fusion protein (H6ScCypB) and enzymic characterization of the purified protein showed that, like ScCypA, ScCypB is a peptidyl-prolyl cis-trans isomerase (PPIase)\. The specific activity and substrate specificity of the enzyme were comparable to that of ScCypA, but it was threefold less sensitive to inhibition by cyclosporin A (CsA)\. In contrast to ScCypA, which is abundant and exists in free and liganded form, ScCypB was 50- to 100-fold less abundant in cytosol-derived protein fractions of S. chrysomallus or Streptomyces lividans, as revealed by Western blot analyses, suggesting a specialized function for this enzyme in the streptomycete cell\. Both sccypB and sccypA were found to be present as single copies in the genome of S\. chrysomallus and hybridized to a single band in chromosomal DNAs of other streptomycetes\. High-level expression of sccypB as well as of sccypA cloned into the expression vector pIJ702 did not produce detectable changes in growth and morphology of S. chrysomallus and S\. lividans\. Calculations of similarities to known cyclophilin sequences and construction of phylogenetic trees indicated that ScCypB and ScCypA are phylogenetically distant from each other\. While ScCypA is clearly related to the eukaryotic cyclophilins, the analyses show the sequence of ScCypB to be the most divergent of all cyclophilin sequences, indicating that it possibly constitutes a cluster by itself.