Cellular Interactome Dynamics during Paclitaxel Treatment

Abstract

Graphical Abstract Highlights d Quantitative cross-linking/mass spectrometry analysis of mitotic inhibitor-treated cells d Cross-links reflect paclitaxel stabilization of microtubules d Drug-specific changes to intermediate and microfilament structures d Paclitaxel treatment alters mitochondrial respiration and ATP synthase structure In Brief Chavez et al. reveal interactome changes in cells treated with mitotic inhibitors using quantitative cross-linking and mass spectrometry. Cross-links reflect interaction/conformational changes specific for drug type and concentration, which are not evident by protein expression levels. Microtubule stabilization, cytoskeletal alteration, and changes to mitochondrial function are visualized in cross-link levels. ratio Drug conc. Quantitative cross-linking Mitotic Inhibitor Protein interaction/structural changes vs SUMMARYCell-cycle inhibitors, including paclitaxel, are among the most widely used and effective cancer therapies. However, several challenges limit the success of paclitaxel, including drug resistance and toxic side effects. Paclitaxel is thought to act primarily by stabilizing microtubules, locking cells in a mitotic state. However, the resulting cytotoxicity and tumor shrinkage rates observed cannot be fully explained by this mechanism alone. Here we apply quantitative chemical cross-linking with mass spectrometry analysis to paclitaxel-treated cells. Our results provide large-scale measurements of relative protein levels and, perhaps more importantly, changes to protein conformations and interactions that occur upon paclitaxel treatment. Drug concentrationdependent changes are revealed in known drug targets including tubulins, as well as many other proteins and protein complexes involved in apoptotic signaling and cellular homeostasis. As such, this study provides insight into systems-level changes to protein structures and interactions that occur with paclitaxel treatment.

Reliable identification of protein-protein interactions by crosslinking mass spectrometry

“…There have been several recent advances in enrichment and detection of crosslinked peptides by Crosslinking MS that suggest it will soon be able to map large portions of the cellular interactome in a single experiment 3 , 13 , 25 . This will open the door to detecting changes in these interactomes in different cellular states by quantification of the abundances of the detected crosslinks 2 , 26 . All of these advances require correctly controlled FDR to produce results that can be relied upon.…”

“…rosslinking mass spectrometry (Crosslinking MS) has become a key technology for understanding the architecture of multi-protein complexes by providing distance restraints between protein residues 1 . These studies are typically performed on purified complexes, but in recent years pioneering studies have used Crosslinking MS to study the topology of PPIs in more complex systems, such as cell lysates, organelles or whole cells [2][3][4][5][6][7][8][9][10][11][12][13] . Crosslinking MS is therefore emerging as a technique for mapping PPIs alongside existing tools, such as two-hybrid screens, affinity purification, proximity labelling techniques and co-fractionation studies.…”

Crosslinking mass spectrometry: A link between structural biology and systems biology

“…Since XL‐MS can be performed with complex samples, such as, living cells, this technique offers the unique ability to visualize large‐scale conformational and protein interaction changes that can occur during perturbation 19,78 . For example, quantitative crosslinking with mass spectrometry (qXL‐MS) enables the assessment of interactome changes in control and drug treated cells 37,79,80 . As with traditional quantitative proteomics, qXL‐MS utilizes either stable isotope labeling or label‐free quantitation methods.…”

“…Introduction of MS-cleavable bond(s) into a crosslinker spacer chain using collisions (PIR, 26 DSBU, 27 and DSSO 28 ), photons (IRCX 29 ), or electrons (DEST 30 ) allows release of linear peptides in gas phase for further fragmentation. Crosslinker with MS-cleavable bond(s) has been an enabling factor for advancing XL-MS for protein structural/interaction studies at proteome-wide scale, including whole cell lysate, [31][32][33][34] living cells, 28,[35][36][37] functional organelle, [38][39][40][41] tissues and organs. 18,42 Incorporation of two MS-cleavable bonds has introduced a novel class of crosslinker molecules named Protein Interaction Reporters (PIR), 26 in which the release of the middle spacer section upon cleavage of both labile bonds can produce reporter ions that serve to readily identify tandem spectra containing crosslinked peptides.…”

Evaluation of the Biological Activity of Folic Acid-Modified Paclitaxel-Loaded Gold Nanoparticles

“…[16][17][18][19] Regrettably, PTX has many toxic side effects such as myelosuppression, neurotoxicity, cardiotoxicity, joint or muscle pain, liver toxicity, and renal toxicity. [20][21][22] Moreover, it is sparingly water-soluble and therefore has limited application as an intravenous injection. 23 Luckily, AuNPs with good water solubility and targeting can reduce the side effects of such drugs and improve their efficacy.…”