Mammalian target of rapamycin (mTOR)
signaling is a core pathway
in cellular metabolism, and control of the mTOR pathway by rapamycin
shows potential for the treatment of metabolic diseases. In this study,
we employed a new proximity biotin-labeling method using promiscuous
biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced
interactome on the FK506-rapamycin binding (FRB) domain in living
cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA
in the presence of rapamycin. Immunoprecipitation and immunofluorescence
experiments confirmed that endogenous FKBP25 has a rapamycin-induced
physical interaction with the FRB domain. Furthermore, the crystal
structure of the ternary complex of FRB–rapamycin–FKBP25
was determined at 1.67-Å resolution. In this crystal structure
we found that the conformational changes of FRB generate a hole where
there is a methionine-rich space, and covalent metalloid coordination
was observed at C2085 of FRB located at the bottom of the hole. Our
results imply that FKBP25 might have a unique physiological role related
to metallomics in mTOR signaling.
Alkyl halides are potentially mutagenic carcinogens. However, no efficient fluorescent sensor for alkyl halide detection in human-derived samples has been developed to date. Herein, we report a new protein-based fluorescent sensor for alkyl halides. Analysis of the HaloTag holo-crystal structure with its covalently attached ligand revealed an unexpected cavity, allowing for the design of a new fluorogenic ligand. This ligand showed the highest fluorescence response (300-fold) and fastest binding kinetics (t < 150 s) to a HaloTag mutant (M175P) protein. This protein-based sensor system was effectively used to detect alkyl halides in human serum and monitor real-time protein alkylation.
Heat shock protein 90 (Hsp90) family proteins are molecular chaperones that modulate the functions of various substrate proteins (clients) implicated in pro-tumorigenic pathways. In this study, the mitochondria-targeted antioxidant mitoquinone (MitoQ) was identified as a potent inhibitor of mitochondrial Hsp90, known as a tumor necrosis factor receptor-associated protein 1 (TRAP1). Structural analyses revealed an asymmetric bipartite interaction between MitoQ and the previously unrecognized drug binding sites located in the middle domain of TRAP1, believed to be a client binding region. MitoQ effectively competed with TRAP1 clients, and MitoQ treatment facilitated the identification of 103 TRAP1-interacting mitochondrial proteins in cancer cells. MitoQ and its redox-crippled SB-U014/SB-U015 exhibited more potent anticancer activity in vitro and in vivo than previously reported mitochondria-targeted TRAP1 inhibitors. The findings indicate that targeting the client binding site of Hsp90 family proteins offers a novel strategy for the development of potent anticancer drugs.
Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.
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