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| 2. |
- Bestas, Burcu, et al.
(författare)
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Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
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Tidskriftsartikel (refereegranskat)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
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| 3. |
- Gustafsson Sfetcovici, Manuela O
(författare)
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Characterization of ankyrin repeat domain 54 (ANKRD54) and its role on the regulation and subcellular localization of Bruton’s tyrosine kinase (BTK)
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bruton's tyrosine kinase (BTK) is an important cytoplasmic signaling protein, where the kinase activity plays a pivotal role in the development, proliferation and differentiation of B-cell lineages. Ankyrin repeat domain 54 (ANKRD54) is a nuclear-resident adaptor protein, where the ankyrin domain repeats are critical for specific protein-protein interaction, while the NLS and NES motifs control the nucleo-cytoplasmic shuttling ability. We have identified and characterized ANKRD54 as a novel functional (paper I), interaction-partner for BTK using proteomics analysis. ANKRD54 is the first protein identified that specially influences the nuclear export of both BTK and TXK/RLK, in a Crm-1 dependent manner. Further, we mapped the interaction site to the C -terminus of BTK-SH3 domain, by using a synthetic peptide of BTK, covering the following region: C- ARDKNGQEEGYIPSNYVTEAEDS. In addition, tyrosine phosphorylation of BTK was investigated in the presence of increased amount of ANKRD54 and selectively the phosphorylation of BTK was down regulated. We have presented a second novel interaction-partner and regulator of BTK (paper II), the 14-3-3 ζ protein, which is also identified by proteomics strategy. In this work, we have mapped the interaction sites on BTK to phospho-serine pS51 in the (RGRRGpS)-motif in the PH-domain and phospho-threonine pT495 in the (RHRFQpT)-motif in the kinase domain. Additionally, a newly characterized 14-3-3 inhibitor (BV02) interfered binding with BTK and siRNA knockdown of 14-3-3ζ increased the nuclear translocation of BTK, while overexpression of 14-3-3ζ resulted in accumulation of BTK in the perinuclear region. We have generated single ankryin domain deletions of ANKRD54 and subsequently characterized their binding capacity and also their influence on the sub-cellular localization of BTK (paper III). In this work, we report that three out of four ankyrin repeats are required for the interaction and nucleo-cytoplasmic shuttling of BTK. Inhibition of Crm-1 nuclear export pathway influences differently the nuclear shuttling; rapid-ANKRD54 versus slow-BTK nuclear accumulation. Furthermore, we have determined that the interaction between BTK and ANKRD54 establishes in the nuclear compartment. We have classified ANKRD54 as a prime interactor to the SH3-domain of BTK (paper IV). In this study, we utilized a screening strategy based on phage display libraries of the complete human “SH3-domainome” as a possible binding-target for ANKRD54. The aim is to identify the target spectrum and specificity of ANKRD54 for SH3 domain library, containing all the 296 human SH3 domains. The novel finding is that BTK is not only binding to ANKRD54, but also stands out as the preferred interactor, being highly dominant over all other human SH3 domains. However, other lower colony-score candidates for SH3-domain interactions were found, but without any further in vivo/in vitro validation.
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| 4. |
- Li, Chen, et al.
(författare)
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Cutaneous squamous cell carcinoma-derived extracellular vesicles exert an oncogenic role by activating cancer-associated fibroblasts
- 2023
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Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFβ signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.
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| 5. |
- Sassi, Atfa, et al.
(författare)
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Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels
- 2014
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Ingår i: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749 .- 1097-6825. ; 133:5, s. 1410-U681
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. Objective: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. Methods: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. Results: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p. Glu340del and p. Leu83Ser). A third homozygous mutation (p. Asp502Tyr) and the p. Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. Conclusion: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.
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| 6. |
- Speliotes, Elizabeth K., et al.
(författare)
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Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index
- 2010
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 42:11, s. 937-948
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Tidskriftsartikel (refereegranskat)abstract
- Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ~2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10−8), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.
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