China
Africa
Explore chapters and articles related to this topic
Imaging Cellular Networks and Protein-Protein Interactions In Vivo
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Snehal Naik, Britney L. Moss, David Piwnica-Worms, Andrea Pichler-Wallace
One intein-mediated split-FLuc reporter system is based on DnaE, a naturally split intein derived from a strain of Cyanobacterium synechocystis that can be reconstituted in trans to ligate N and C terminals of exteins (23–25). Two interacting proteins were fused, one to a fusion protein consisting of the N-terminal of DnaE and an N-terminal fragment of firefly luciferase (N-FLuc) and the other to a fusion protein consisting of the C-terminal of DnaE and a C-terminal fragment of firefly luciferase (C-FLuc). An interaction between the two proteins of interest allows for formation of an intact DnaE, leading to protein splicing and formation of a mature FLuc. This technique was used to study insulin-stimulated phosphorylation of insulin receptor substrate 1 (IRS-1) and its target, PI3-kinase-derived SH2N (p85 subunit of phosphatidylinositol 3-kinase) domain, in living cells (23). The next generation of split-intein-mediated luciferases used split Renilla luciferase (RLuc), instead of FLuc (25), to study ligand-induced protein trafficking into the nucleus both via cell-based in vitro assays and in vivo imaging of mice. Specifically, translocation of the androgen receptor (AR) from the cytosol to the nucleus was monitored upon stimulation with 5α-dihydrotestosterone (DHT) by fusing the N-terminus of RLuc to the N-terminus of DnaE and a nuclear localization sequence (NLS). The AR was fused to the C-terminus of RLuc, which in turn was fused in frame to the C-terminus of DnaE. Upon DHT stimulation, AR would translocate to the nucleus, allowing the split inteins to associate, and resulting in formation of an active RLuc protein. Addition of DHT showed two- to eightfold induction in signal in a concentration-dependent manner, which could be inhibited using antagonists. The potential utility of this bioluminescence-based technique for high-throughput screening of chemicals was simulated using 13 different chemicals and its in vivo application was demonstrated using either subcutaneous (s.c.) or intracranial (i.c.) implantation of cells cotransfected with each split fusion protein. Using this technique in combination with animals that are genetically engineered to express split fragments in specific tissues, it may be possible to monitor translocations of proteins of interest in target tissues as well as monitor activity of agonist and antagonist drugs mediating those translocations specifically in various organs.
How valuable can proteogenomics be in clinical breast cancer research?
Published in Expert Review of Proteomics, 2023
Anh M. Tran-Huynh, Matthew V. Holt, Meenakshi Anurag
Marchi et. al (2021) incorporated a new workflow into a BC cohort (24 tumors) that provided high proteome depth and quantitative accuracy from a single-shot analysis [25]. This study used proteogenomics to detect understudied tumor molecular features such as protein splicing isoforms and mutational-defined single amino acid variants. Differential expression of variant-specific transcripts and proteins were uncovered between ER-positive and ER-negative subtypes, suggesting distinct functions for different isoforms of the same protein. Additionally, their group also applied proteogenomics to uncover the relationship between hormonal receptors status and proliferation levels resulting from somatic alterations in context of tumor evolution [26]. Recently, Hari et. al (2022) initiated a new proteogenomics pipeline to re-analyze TCGA BRCA samples and CPTAC MS-based proteomic data by de novo transcript assembly methods (Trinity) that had greater strength of identifying novel transcripts [27]. They successfully identified 8 novel peptide sequences strongly associated with patient survival.
Locked and loaded: engineering and arming oncolytic adenoviruses to enhance anti-tumor immune responses
Published in Expert Opinion on Biological Therapy, 2022
Insertion of splice acceptor (SA) sites hijack intrinsic viral RNA splicing machinery to stitch foreign cDNAs into viral transcripts to allow transgene expression. This approach is similar to replacing a viral ORF but avoids deleting what may be an important viral protein. This approach has the great advantage of minimizing insert size because SA sequences can be as small as 30–50 bp. This strategy has been reported by using SA sites derived from Ad5 pIIIa(IIIaSA) [83], Ad40 long fiber (40SA) [84], Ad41 long fiber (41SA) [85], SV40 large T antigen [86] and beta globulin gene [87]. It is critical to check and optimize the transgene sequence to avoid potential inhibitory splicing cis-elements within transgene as they can interfere with viral protein splicing and transgene expression [81]. These elements usually need to be placed after stop codons but before polyA elements. These approaches are best made at the end of transcriptional units to avoid deranging normal viral splicing.
A monoclonal antibody with broad specificity for the ligands of insulin B:9-23 reactive T cells prevents spontaneous type 1 diabetes in mice
Published in mAbs, 2020
Joseph Ray Cepeda, Nitin S Sekhar, Junying Han, Wei Xiong, Ningyan Zhang, Liping Yu, Shaodong Dai, Howard W. Davidson, John W. Kappler, Zhiqiang An, Li Zhang
Insulin, the signature product of pancreatic ß cells, comprises ~10% of the total protein in the cell,32 and is generally regarded as a primary target of islet autoimmunity in both animal models and human.33 Indeed, polymorphisms in the INS gene that decrease thymic expression are the second highest genetic risk factor for developing T1D.34 Thus, (pro)insulin is a logical target of efforts to develop an effective ASI for T1D.31 Although most research has focused on the native protein, there is growing awareness that post-translational modifications that create neo-antigens within the target tissue may be of particular importance in breaking immune tolerance in T1D.35 One such mechanism is protein-splicing, which we believe is critical to the generation of the highly pathogenic epitopes from the insulin B chain8 that are recognized by mAb757. As discussed above, B:9–23 reactive CD4+ T cells isolated from the islets of NOD mice can be segregated into two types termed “type A” and “type B.” The fact that mAb757 is able to readily block activation of both specificities, but has minimal affinity for I-Ag7 loaded with irrelevant peptides, appears inconsistent with the hypothesis that the two T cell types recognize the same peptide bound in different registers as originally proposed.14 Rather it strongly supports our alternate proposal that “type A” and “type B” cells each recognize complexes in which the peptide is bound in the same register, but has different post-translational modifications to the carboxy-terminus.8 Given that the relative importance of the two specificities remains uncertain, and may vary at different stages of disease, we reasoned that a broad specificity antibody capable of blocking activation of both T cell types would have greater therapeutic potential than one that shows a significant preference for either “type A” or “type B,” such as mAb287. The results described above support this concept.