Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.

Inhibitors of ERp44, PDIA1, and AGR2 induce disulfide-mediated oligomerization of Death Receptors 4 and 5 and cancer cell death.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Homo- and Heteropolymetallic Complexes of the Hybrid, Ambidentate N-Heterocyclic Carbene Ligand IMes-acac.

The anionic 5-acetylimidazol-2-ylidene-4-olate 1 -, named as "IMes-acac", is composed of fused diaminocarbene and acetylacetonato units in the same IMes-based imidazolyl ring. The bifunctional compound 1 - is shown to act as an effective, ditopic bridging ligand for transition metal centers. Several new complexes supported by this ligand were prepared, including the complex [RuCl(p-Cym)(κ2 O,O-1·H)](BF4) (2), which can be regarded as a metallated imidazolium salt, the homobimetallic complex [((COD)Rh)(RhCl(COD))(µ-1κ2 O,O:2κ1 C-1)] (4), the heterobimetallic complexes [((p-Cym)ClRu)(RhCl(COD))(µ-1κ2 O,O:2κ1 C-1)] (3), [((p-Cym)ClRu)(RhCl(CO)2)(µ-1κ2 O,O:2κ1 C-1)] (5), [((p-Cym)ClRu)(Cu(IPr))(µ-1κ2 O,O:2κ1 C-1)] (9), the anionic homoleptic Cu(I) complexes [Cu(κ1 C-1)2]K ([10]K) and [Cu(κ1 C-1)2](NEt4) ([10](NEt4)), and the heterotrimetallic complex [((p-Cym)RuCl)2(Cu)(µ-1κ2 O,O:3κ1 C-1)(µ-2κ2 O,O:3κ1 C-1)](PF6) (11). Preliminary studies on the possible preparation of supramolecular metallopolymers and electrochemical studies on the series of complexes are also reported.