Showing papers by "Shao En Ong published in 2023"

Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells

Abstract: Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing's syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.1,2 For complete details on the use and execution of this protocol, please refer to Omar et al. (2022).3.

Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ

Abstract: Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

Pos1008 investigating antigen specific t cells and their citrullinated protein targets in synovial tissue

Abstract: Rheumatoid arthritis (RA) is a T cell mediated autoimmune disease in which citrullinated self-antigens are recognized by anti-citrullinated protein antibodies (ACPA) and T cells. To date, the majority of T cell studies have been performed using peripheral blood and have focused on well-documented ACPA targets. Studies of disease-affected tissue are needed to confirm and extend observations that have been made through study of peripheral blood T cells.T cell subsets and targets that have not been observed in peripheral blood are likely to comprise an important (but as yet, understudied) component of the antigen specific responses that underlie RA. We sought to generate novel insights about T cell phenotypes and antigenic targets by performing multicolor flow cytometry analysis and HLA peptidomics studies of synovial tissue samples from subjects with RA.Synovial tissue was obtained from 7 subjects with seropositive RA, 4 subjects with seronegative RA, and 8 subjects with osteoarthritis, all of whom had undergone arthroplasty procedures. Synovial cell suspensions were obtained from tissue through mincing, digestion in collagenase I, and filtration. These were subjected to multicolor flow cytometry analysis to gain insights about the lymphocyte and non-lymphocyte cell subsets present. After confirming leukocyte antigen (HLA) protein expression by flow cytometry, additional tissue was solubilized in lysis buffer and HLA class I and HLA-DR complexes were captured (separately) on affinity columns. HLA-bound peptides were eluted, concentrated, and peptide spectra were identified by LC-MS/MS analysis. The resulting datasets were assigned sequences by searching against a human protein database. Mass shifts associated with each assigned sequence were utilized to identify post-translational modifications – most notably citrullination of native arginine residues. Comprehensive libraries of the HLA-Class I- and HLA-DR-bound peptides from each individual were imported into a custom database, which was then used to catalogue the most prevalent self-proteins for each patient type. T cell assays were then performed to demonstrate the immunogenicity of novel targets and probe T cell antigen specificity in tissue.Flow cytometry analysis of synovial tissue derived cells demonstrated that fibroblasts (including fibroblast-like synoviocytes), monocytes, and B cells were all present in tissue. In particular, fibroblasts and monocytes showed evidence of inflammation, including upregulated levels of HLA-DR expression. Similar numbers of CD4+ and CD8+ T cells were present in tissue, with phenotypes that included various memory subsets but essentially no naïve cells. In comparison with peripheral blood, T cells from synovial tissue showed evidence of recent activation, expressing higher levels of CD95, CD71, PD-1, ICOS, and CD69. The most prevalent self-proteins in the HLA-DR-bound peptidome from the synovial tissue of RA subjects included expected targets such as vimentin, alpha-enolase, fibrinogen, collagen, histones, and BIP but also contained novel targets such as fibronectin, gelsolin, and proteoglycan 4. Prevalent self-proteins in the HLA-Class I-bound peptidome included well-studied CD4+ T cell targets such as vimentin, alpha-enolase, and collagen but also contained more novel targets such as caspase-14, stromelysin-1, and filamin-A. T cell assays supported the immunogenicity of expected targets and the new candidate antigen gelsolin. In particular, a comparatively robust population of aggrecan specific T cells was present in tissue.Our findings demonstrate that flow cytometry and HLA peptidomic analysis of synovial tissue can provide novel insights about the phenotype and antigen specificity of T cells in RA. Further characterization of T cell response in tissue, including those that recognize novel antigens, has the potential to provide important new insights about the character of antigen specific T cell responses that promote the development of RA.NIL.NIL.Eddie A. James Grant/research support from: Janssen, BMS, Pfizer, Novartis, Sanofi, Cliff Rims: None declared, Sylvia Posso: None declared, Jeffrey Carlin: None declared, William Kwok Grant/research support from: BMS, Shao-En Ong: None declared, Jane Buckner Consultant of: BMS, Grant/research support from: Janssen, BMS.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma

Abstract: The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.