DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate γ-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with γ-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of γ-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.
Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia. 1 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany. 2 Department of Hematology/Oncology, University Medical School, Frankfurt, Germany. 3 Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany. 4 Molecular Epidemiology Group, German Cancer Research Center, Heidelberg, Germany. 5 University Women's Clinic, Division Molecular Biology of Breast Cancer, Heidelberg, Germany. 6 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany. 7 Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center and Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany. 7 Pediatric Hematology, Oncology and Hemostaseology, University Medical School, Frankfurt, Germany. 8 Department of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany. 9 Department of Hematology, Oncology and Transfusion Medicine, Charité, Campus Benjamin Franklin, Berlin, Germany. 10 Institute of Pathology, Heinrich-Heine University, Düsseldorf, Germany. 11 EUFETS AG, Idar-Oberstein, Germany. 12 Centre for Immunodeficiency, UCL Institute of Child Health, and Great Ormond Street Hospital for Children NHS Trust London, UK. 13 Division of Immunology/Hematology, University Children's Hospital Zurich, Zurich, Switzerland. 15 These authors contributed equally to this work. a r t i c l e sThe subject received daily granulocyte colonystimulating factor (G-CSF) support (5 µg per kg body weight per day) from months 18 to 20 and months 24 to 26, as well as multiple red blood and platelet transfusions. Following a dental abscess and a febrile episode requiring antibiotic and antimycotic treatment, subject 1 was noted to have extensive splenomegaly and underwent splenectomy at month 25 to avoid spontaneous rupture. Histopathological examination of the spleen revealed extramedullary hematopoiesis and siderosis in the red pulp, without signs of dys...
The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.
The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.
We looked for membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitinspeci¢c processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at membranes. For each of the Ubps examined, a certain fraction was found to be membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the membrane fraction suggesting that this protein performs a major function at membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively membrane associated. By cell fractionation and immuno£uorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast outer mitochondrial membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral membrane protein of the outer mitochondrial membrane with an N in^Cout orientation. Phenotypic characterization of the v vubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria. ß
Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment. INTRODUCTIONMany cellular proteins are modified by the attachment of the 76-amino acid polypeptide ubiquitin (Hochstrasser, 1996;Hershko and Ciechanover, 1998). The main role of ubiquitination is to target proteins for degradation either directly by acting as a degradation signal that is recognized by the 26S proteasome or indirectly by sorting membrane proteins into the lysosomal/vacuolar degradation pathway (Hicke, 1999). Ubiquitination of substrate proteins, which occurs by a cascade of enzymatic reactions, is reversible. Ubiquitin can again be removed from proteins by deubiquitinating enzymes (Dubs). A large number of Dubs have been identified in various organisms that are either cysteine proteases or metalloproteases (Verma et al., 2002; Yao and Cohen, 2002). The more classical cysteine proteases can again be divided into two groups: the ubiquitin C-terminal hydrolases (Uchs) that preferentially cleave ubiquitin from peptides and small adducts (e.g., Yuh1 in yeast) and the extremely divergent family of ubiquitin-specific processing proteases (Ubps) that cleave ubiquitin from protein substrates (Hochstrasser, 1996). Of the 17 cysteine-protease-type Dubs in yeast 16 belong to the Ubp class.The degree of ubiquitination seems to be mainly regulated at the level of ubiquitin attachment. However, evidence is accumulating that deubiquitination also can be important for the regulation of ubiquitination levels. Evidence has been presented that the deubiquitinating enzyme Fat facets (Faf), which is involved in eye development in Drosophila, acts as a substrate-specific regulator of ubiquitination of the...
Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR-specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv (225) Epidermal growth factor receptor (EGFR, ErbB) belongs to the family of ErbB receptor tyrosine kinases that also includes the closely related ErbB2 (HER2/Neu), ErbB3 (HER3) and ErbB4 (HER4) molecules.1 These type I transmembrane proteins share a common molecular architecture characterized by a glycosylated extracellular domain to which peptide ligands bind, a single a-helical transmembrane region and a cytosolic domain with tyrosine kinase activity. The EGFR extracellular domain is structurally separated into four subdomains. Subdomains I and III together provide the binding interface for ligands of the EGF-like growth factor family. Ligand binding stabilizes a dimerization-competent state of the receptor by preventing an inhibitory intramolecular contact between subdomains II and IV, thereby exposing a dimerization interface in subdomain II that facilitates the formation of receptor homodimers or heterodimers with other ErbB family members.2 Receptor dimerization stimulates the intrinsic tyrosine kinase activity, which results in autophosphorylation of specific tyrosine residues within the C-terminal tail. These phosphotyrosine residues serve as docking sites for intracellular substrates and adapter proteins that initiate signaling cascades promoting cell survival, migration and proliferation.
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