Cleavable Linkers in Chemical Proteomics Applications

The discovery of the protein targets of small molecule probes is a crucial aspect of activity-based protein profiling and chemical biology. Mass spectrometry is the primary method for target identification, and in the last decade, cleavable linkers have become a popular strategy to facilitate protein enrichment and identification. In this chapter, we provide an overview of cleavable linkers used in chemical proteomics approaches, discuss their different chemistries, and describe how they aid in protein identification.status: publishe

Circular RNA hsa_circ_0062682 Binds to YBX1 and Promotes Oncogenesis in Hepatocellular Carcinoma

Circular RNAs (circRNAs) have been shown to play an important role in the pathogenesis of hepatocellular carcinoma (HCC). By implementing available transcriptomic analyses of HCC patients, we identified an upregulated circRNA hsa_circ_0062682. Stable perturbations of hsa_circ_0062682 in Huh-7 and SNU-449 cell lines influenced colony formation, migration, cell proliferation, sorafenib sensitivity, and additionally induced morphological changes in cell lines, indicating an important role of hsa_circ_0062682 in oncogenesis. Pathway enrichment analysis and gene set enrichment analysis of the transcriptome data from hsa_circ_0062682 knockdown explained the observed phenotypes and exposed transcription factors E2F1, Sp1, HIF-1α, and NFκB1 as potential downstream targets. Biotinylated oligonucleotide pulldown combined with proteomic analyses identified protein interaction partners of which YBX1, a known oncogene, was confirmed by RNA immunoprecipitation. Furthermore, we discovered a complex cell-type-specific phenotype in response to the oncogenic potential of hsa_circ_0062682. This finding is in line with different classes of HCC tumours, and more studies are needed to shed a light on the molecular complexity of liver cancer

Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

<div><p>Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.</p> </div

An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy

Identification of profilin 1 as a substrate for cathepsin X carboxypeptidase activity.

<p>(<b>A</b>) Control versus AMS-36 treated sample is shown after 2D electrophoresis. The spot marked with arrow was identified as human profilin 1. (<b>B and C</b>) The C-terminal of profilin 1 (SHLRRSQY) (800 µM) was digested with recombinant cathepsin X (4.62 µM) at 37°C for 30 minutes and separated on a C18 Gemini column (5 µm, 110 Å, 150×4.6 mm) (Phenomenex). (<b>B</b>) 5 additional peaks, named peaks 2 to 6, were detected besides the original octapeptide (black line). The octapeptide control without enzyme is shown in red. (<b>C</b>) Q-TOF Premier mass spectrometry analysis of each peak showed the presence of 3 to 7 amino acid long peptides, all shortened by 1 amino acid from the C- terminal. (<b>D</b>) Profilin 1 (1 µg/µl; Abcam) was digested with recombinant cathepsin X (46.2 µM) at 37°C for several hours and the digestion product detected with mass spectrometry. A new peak was detected with molecular mass matching the mass of profilin 1 without the last amino acid residue Tyr.</p

Cathepsin X in MCF-7, MDA-MB-231 and PC-3 cells.

<p>(<b>A</b>) Activity was measured in cell lysates using cathepsin X specific substrate Abz-FEK(Dnp)-OH. Mean values of 5 independent experiments are shown. ***P<0,001 (<b>B</b>) The amount of cathepsin X (ng/ml) in different cell lines was determined with ELISA. Mean values of 2 (MCF-7 and MDA-MB-231) or 4 (PC-3) independent experiments are shown.</p

Cathepsin X increases migration, adhesion and invasion of cancer cells.

<p>Migration (A, B), adhesion (C) and invasion (D) assays were done using xCELLigence System. (<b>A and B</b>) Diagrams show a slope (cell index vs. time) of cells that migrated from the upper to the lower chamber. Cells migrated in the presence of DMSO (red line on graph) or 10 µM inhibitor of cathepsin X (green line on graph) (A) or cells, transfected with control (red line on graph) or cathepsin X specific siRNA (green line on graph) were used (B). (<b>C</b>) Diagram shows a slope for cells transfected with a control (red line on graph) or cathepsin X specific siRNA (green line on graph), that adhered to fibronectin (10 µg/ml). (<b>D</b>) Diagram shows a slope for cells transfected with a control (red line on graph) or cathepsin X specific siRNA (green line on graph) that invaded through Matrigel from the upper to the lower chamber. ***P≤0.01; ***P<0,001. Graphs show real-time curves of cell index (CI) as a function of time. Vertical lines represent the start and end of time intervals within which corresponding diagrams are calculated. Four (A), eight (B), four (C) and five (D) biological repeats were performed.</p