Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 989-989 ◽  
Author(s):  
Abdullah M. Ali ◽  
Thiyam R. Singh ◽  
Ruhikanta A. Meetei
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Affinity Purification ◽  
Gene Products ◽  
Core Complex ◽  
Purification Method

Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.

Elucidating the Molecular Architecture of the FA-Core Complex Utilizing Biochemical Purification Approach.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1683-1683
Author(s):  
Abdullah M. Ali ◽  
Changhu Du ◽  
Thiyam R. Singh ◽  
Ruhikanta A. Meetei
Keyword(s):  
Cell Lines ◽  
Affinity Purification ◽  
Bone Marrow Failure ◽  
Chromosome Instability ◽  
Functional Characterization ◽  
Molecular Architecture ◽  
Core Complex ◽  
Biochemical Purification ◽  
Purification Method ◽  
Hela Cell Lines

Abstract FA is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. FA is genetically heterogeneous, represented by at least thirteen different complementation groups. The genes corresponding to groups A, B, C, D1 (BRCA2), D2, E, F, G, I, J, L, M and N have been cloned. The Fanconi proteins FANC -A, -B, -C, -E, -F, -G, -L, -M along with another newly discovered protein FAAP100 forms a core complex (CC) in the nucleus that is required for the monoubiquitination of two other Fanconi proteins FANCD2 and FANCI. In addition to their presence in nucleus, most of the Fanconi proteins are also found in the cytosol however their role in cytosol is not clear. Also, whether the FA-core complex proteins exist as a single complex or sub-complexes in the cytosol is not clear. The goals of this study are to develop a method of purification of FA core complex from nuclear and cytosol identify individual components of cytosolic and nuclear complex(s) of the FA proteins. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCC, FANCG, FANCL and FAAP100 proteins; we have established four different HeLa cell lines; HeLa-HF-FANCC, HeLa-HF-FANCG, HeLa-HF-FANCL and HeLa-HF-FAAP100, stably expressing HF-FANCC, HF-FANCG, HF-FANCL and HF-FAAP100 recombinant proteins respectively. Affinity purification was carried out to isolate the complexes from the cytosol and nuclear extracts prepared from stable cell lines. The polypeptides purified from the complexes were identified by mass spectrometry. The results suggest that, in addition to the existence of the FA-core complex containing FANC -A, -B, -C, -E, -F, -G, -L and 100, the FA proteins also exists as sub-complexes in the cytosol. FANCL was found to form a sub-complex with FANCB and FAAP100 in cytosol. Also FANCC, a predominantly cytosolic protein was found to form a sub-complex with FANCE in cytosol. We found several novel proteins that interact with FA proteins.

scholarly journals Mass Spectrometry-Based Proteomic Characterization of Cutaneous Melanoma Ectosomes Reveals the Presence of Cancer-Related Molecules

2020 ◽  
Vol 21 (8) ◽  
pp. 2934 ◽  
Author(s):  
Magdalena Surman ◽  
Sylwia Kędracka-Krok ◽  
Dorota Hoja-Łukowicz ◽  
Urszula Jankowska ◽  
Anna Drożdż ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Content ◽  
Cell Lines ◽  
Cutaneous Melanoma ◽  
Epithelial Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Starting Point ◽  
Novel Biomarkers

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC–MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial–mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1975-1989 ◽  
Author(s):  
T. Nilsson ◽  
C. Rabouille ◽  
N. Hui ◽  
R. Watson ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Cell Lines ◽  
Hela Cells ◽  
Kin Recognition ◽  
Dramatic Effect ◽  
Golgi Stack ◽  
Stable Cell Lines ◽  
Membrane Spanning ◽  
Necessary And Sufficient

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

scholarly journals Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5056-5056 ◽  
Author(s):  
Julia Fremerey ◽  
Pavel Morozov ◽  
Cindy Meyer ◽  
Aitor Garzia ◽  
Marianna Teplova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Binding Properties ◽  
Expression Levels ◽  
Rna Targets ◽  
Polymerase I

Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

The growing role of structural mass spectrometry in the discovery and development of therapeutic antibodies

The Analyst ◽  
2018 ◽  
Vol 143 (11) ◽  
pp. 2459-2468 ◽  
Author(s):  
Yuwei Tian ◽  
Brandon T. Ruotolo
Keyword(s):  
Mass Spectrometry ◽  
State Of The Art ◽  
Therapeutic Antibodies ◽  
Critical Importance ◽  
Current State ◽  
Measurement Science ◽  
Structural Mass Spectrometry ◽  
Structural Mass

The comprehensive structural characterization of therapeutic antibodies is of critical importance for the successful discovery and development of such biopharmaceuticals, yet poses many challenges to modern measurement science. Here, we review the current state-of-the-art mass spectrometry technologies focusing on the characterization of antibody-based therapeutics.

Genome Wide DNA Methylation Profiling In Patients with Multiple Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3622-3622
Author(s):  
Yang Liu ◽  
Shenghua Duan ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
Abdel Kareem A. Azab ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
Promoter Region ◽  
Genomic Dna ◽  
Research Funding ◽  
Plasma Cells ◽  
Genome Wide

Abstract Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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