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Identification of novel putative regulators of the major apoptotic nuclease DNA Fragmentation Factor

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Hanus J., Kalinowska-Herok M., Widlak P.
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2010
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vol. 57
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issue 4
521-527
EN
Yeast two- and three-hybrid systems were used to screen cDNA libraries from HeLa cells and human brain tissue to identify novel protein partners of DNA Fragmentation Factor, the major apoptotic nuclease. The two-hybrid system revealed the DFF45 inhibitory subunit of the nuclease as the only identified partner of the DFF40 catalytic subunit. Similar analysis revealed several protein candidates that potentially interact with the DFF45 subunit: FBXO28, FOSL1, PGK1, PCNT, FHL1 and GFAP. Recombinant GFAP protected DFF45 against cleavage with caspase-3 and prevented activation of the DFF nuclease in vitro. In addition, three-hybrid system results revealed a putative novel protein partner of the DFF40-DFF45 heterodimer. The candidate cDNA contained two open reading frames that mapped to an intron of the GBF1 gene. Products of the candidate cDNA derived from a cell-free transcription/translation system inhibited DNA cleavage by recombinant caspase-activated DFF. This putative partner of DFF may have functional importance in regulating the apoptotic response because its RNAi silencing facilitated cleavage of the DFF45 inhibitor subunit and affected chromatin fragmentation in HeLa cells undergoing apoptosis. This hypothetical protein, named DRIG based on an acronym specifying its genomic location, could be a novel factor involved in regulation of DFF40 apoptotic nuclease.
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Panel of serum metabolites discriminates cancer patients and healthy participants of lung cancer screening - a pilot study

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Roś-Mazurczyk M., Wojakowska A., Marczak Ł., Polański K., Pietrowska M., Polanska J., Dziadziuszko R., Jassem J., Rzyman W., Widlak P.
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2017
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vol. 64
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issue 3
513-518
EN
Introduction. Blood biomarkers may support early diagnosis of lung cancer by enabling pre-selection of candidates for computed tomography screening or discrimination between benign and malignant screening-detected nodules. We aimed to identify features of serum metabolome distinguishing individuals with early-detected lung cancer from healthy participants of the lung cancer screening program. Methods. Blood samples were collected in the course of a low-dose computed tomography screening program performed in the Gdansk district (Northern Poland). The analysis included 31 patients with screening-detected lung cancer and the pair-matched group of 92 healthy controls. The gas chromatography coupled to mass spectrometry (GC/MS) approach was used to identify and quantify small metabolites present in serum. Results. There were several metabolites detected in the sera whose abundances discriminated patients with lung cancer from controls. Majority of the differentiating components were downregulated in cancer samples, including amino acids, carboxylic acids and tocopherols, whereas benzaldehyde was the only compound significantly upregulated. A classifier including nine serum metabolites allowed separation of cancer and control samples with 100% sensitivity and 95% specificity. Conclusions. Signature of serum metabolites discriminating between cancer patients and healthy participants of the early lung cancer screening program was identified using a GC/MS metabolomics approach. This signature, though not validated in an independent dataset, deserves further investigation in a larger cohort study.
3
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Direct interaction of Gas41 and Myc encoded by amplified genes in nervous system tumours

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Piccinni E., Chelstowska A., Hanus J., Widlak P., Loreti S., Tata A. M., Augusti-Tocco G., Bianchi M. M., Negri R.
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2011
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vol. 58
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issue 4
529-534
EN
In order to understand better the role of the human Tip60 complex component Gas41, we analysed its expression levels in brain tumours and searched for possible interactors. Two-hybrid screening of a human foetal brain library allowed identification of some molecular interactors of Gas41. Among them we found n-Myc transcription factor. The interaction between Gas41 and n-Myc was validated by pull-down experiments. We showed that Gas41 is able to bind both n-Myc and c-Myc proteins, and that the levels of expression of Gas41 and Myc proteins were similar to each other in such brain tumors as neuroblastomas and glioblastomas. Finally, in order to identify which region of Gas41 is involved in the interaction with Myc proteins, we analysed the ability of Gas41 to substitute for its orthologue Yaf9 in yeast; we showed that the N-terminal portions of the two proteins, containing the YEATS domains, are interchangeable, while the C-terminal portions are species-specific. In fact we found that Gas41 C-terminal portion is required for Myc protein interaction in human.
4
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Ionizing radiation affects protein composition of exosomes secreted in vitro from head and neck squamous cell carcinoma

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Jelonek K., Wojakowska A., Marczak L., Muer A., Tinhofer-Keilholz I., Lysek-Gladysinska M., Widlak P., Pietrowska M.
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2015
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vol. 62
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issue 2
265-272
EN
Exosomes are membrane vesicles of endocytic origin that participate in inter-cellular communication. Environmental and physiological conditions affect composition of secreted exosomes, their abundance and potential influence on recipient cells. Here, we analyzed protein component of exosomes released in vitro from cells exposed to ionizing radiation (2Gy dose) and compared their content with composition of exosomes released from control not irradiated cells. Exosomes secreted from FaDu cells originating from human squamous head and neck cell carcinoma were analyzed using LC-MS/MS approach. We have found that exposure to ionizing radiation resulted in gross changes in exosomal cargo. There were 217 proteins identified in exosomes from control cells and 384 proteins identified in exosomes from irradiated cells, including 148 "common" proteins, 236 proteins detected specifically after irradiation and 69 proteins not detected after irradiation. Among proteins specifically overrepresented in exosomes from irradiated cells were those involved in transcription, translation, protein turnover, cell division and cell signaling. This indicated that exosomal cargo reflected radiation-induced changes in cellular processes like transient suppression of transcription and translation or stress-induced signaling.
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