Constanze Wiek

Mutation of the RAD51C gene in a Fanconi anemia- like disorder

Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination–mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.

DLK-1 as a marker to distinguish unrestricted somatic stem cells and mesenchymal stromal cells in cord blood

In addition to hematopoietic stem cells, cord blood (CB) also contains different nonhematopoietic CD45-, CD34- adherent cell populations: cord blood mesenchymal stromal cells (CB MSC) that behave almost like MSC from bone marrow (BM MSC) and unrestricted somatic stem cells (USSC) that differentiate into cells of all 3 germ layers. Distinguishing between these populations is difficult due to overlapping features such as the immunophenotype or the osteogenic and chondrogenic differentiation pathway. Functional differences in the differentiation potential suggest different developmental stages or different cell populations. Here we demonstrate that the expression of genes and the differentiation toward the adipogenic lineage can discriminate between these 2 populations. USSC, including clonal-derived cells lacking adipogenic differentiation, strongly expressed δ-like 1/preadipocyte factor 1 (DLK-1/PREF1) correlating with high proliferative potential, while CB MSC were characterized by a strong differentiation toward adipocytes correlating with a weak or negative DLK-1/PREF1 expression. Constitutive overexpression of DLK-1/PREF1 in CB MSC resulted in a reduced adipogenic differentiation, whereas silencing of DLK-1 in USSC resulted in adipogenic differentiation.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene

Background Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. Objective Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. Methods Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. Results Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2–M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2–RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA–BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. Conclusions XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.

CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of ω-3-Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331P Variant

Biettis crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

Human Cytomegalovirus Disrupts the Major Histocompatibility Complex Class I Peptide-Loading Complex and Inhibits Tapasin Gene Transcription

ABSTRACT Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8+ T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and β2-microglobulin (β2m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8+ T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMV-infected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.

The centrosome and mitotic spindle apparatus in cancer and senescence

Altered cell division is associated with overproliferation and tumorigenesis, however, mitotic aberrations can also trigger antiproliferative responses leading to postmitotic cell cycle exit. Here, we focus on the role of the centrosome and in particular of centrosomal TACC (transforming acidic coiled coil) proteins in tumorigenesis and cellular senescence. We have compiled recent evidence that inhibition or depletion of various mitotic proteins which take over key roles in centrosome and kinetochore integrity and mitotic checkpoint function is sufficient to activate a p53-p21WAF driven premature senescence phenotype. These findings have direct implications for proliferative tissue homeostasis as well as for cellular and organismal aging.

Adaptation of topoisomerase I paralogs to nuclear and mitochondrial DNA

Topoisomerase I is essential for DNA metabolism in nuclei and mitochondria. In yeast, a single topoisomerase I gene provides for both organelles. In vertebrates, topoisomerase I is divided into nuclear and mitochondrial paralogs (Top1 and Top1mt). To assess the meaning of this gene duplication, we targeted Top1 to mitochondria or Top1mt to nuclei. Overexpression in the fitting organelle served as control. Targeting of Top1 to mitochondria blocked transcription and depleted mitochondrial DNA. This was also seen with catalytically inactive Top1 mutants, but not with Top1mt overexpressed in mitochondria. Targeting of Top1mt to the nucleus revealed that it was much less able to interact with mitotic chromosomes than Top1 overexpressed in the nucleus. Similar experiments with Top1/Top1mt hybrids assigned these functional differences to structural divergences in the DNA-binding core domains. We propose that adaptation of this domain to different chromatin environments in nuclei and mitochondria has driven evolutional development and conservation of organelle-restricted topoisomerase I paralogs in vertebrates.

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.

Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

Peptide-independent stabilization of MHC class I molecules breaches cellular quality control

ABSTRACT The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, Kb-Y84C, in which two &agr;-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. Kb-Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (&bgr;2m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and &bgr;2m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by &bgr;2m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that &bgr;2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.

Bile salt export pump‐reactive antibodies form a polyclonal, multi‐inhibitory response in antibody‐induced bile salt export pump deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)