Nagy, T. M. | bioRxiv Science

bioRxiv2025

Bcl-2 Oligomerizes Bax on the Mitochondrial Membrane Surface Preventing the Initial Stages of Apoptosis

The Bcl-2 family of proteins control mitochondrial outer membrane (MOM) pore formation, crucial to cellular clearance via apoptosis. However, the molecular principles by which opposing family members inhibit, mediate or promote MOM perforation remain elusive. Here, we demonstrate that cell-protecting Bcl-2 directly sequesters cell-killing Bax into a protein-protein complex at the membrane interface preventing Bax forming apoptotic pores. Neutron reflectometry showed Bax association with Bcl-2 occurs through the formation of Bax protein oligomers on the membrane surface. Bax binding to the membrane surface was proportional to the membrane-embedded Bcl-2 suggestive of protein-protein complex formation through both Bcl-2/Bax and Bax/Bax interactions. Bcl-2/Bax sequestration to prevent perforation was observed in membrane models with and without pro-apoptotic cardiolipin present. Our findings shed fundamental new light on the communication of Bcl-2 with its cell-killing relatives at the mitochondrias membraneous exterior to prevent cells from undergoing apoptosis. TeaserMembrane-embedded Bcl-2 sequesters Bax, to prevent the perforation of mitochondrial membranes by Bax which would initiate apoptosis.

biophysics↗

bioRxiv2024

Direct modulation of TRPM8 ion channels by rapamycin and analog macrolide immunosuppressants

Rapamycin (sirolimus), a macrolide compound isolated from the bacterium Streptomyces hygroscopicus, is widely used as oral medication for the prevention of transplant rejection and the treatment of lymphangioleiomyomatosis. It is also incorporated in coronary stent coatings to prevent restenosis and in topical preparations for the treatment of skin disorders. Rapamycins in vivo activities are generally ascribed to its binding to the protein FKBP12, leading to potent inhibition of the mechanistic target of rapamycin kinase (mTOR) by the FKBP12-rapamycin complex. The specific rapamycin-induced interaction between domains from mTOR and FKBP12 is also frequently employed in cell biological research, for rapid chemically-induced protein dimerization strategies. Here we show that rapamycin activates TRPM8, a cation channel expressed in sensory nerve endings that serves as the primary cold sensor in mammals. Using a combination of electrophysiology, Saturation Transfer Triple-Difference (STTD) NMR spectroscopy and molecular docking-based targeted mutagenesis, we demonstrate that rapamycin directly binds to TRPM8. We identify a rapamycin-binding site in the groove between voltage sensor-like domain and the pore domain, distinct from the interaction sites of cooling agents and known TRPM8 agonists menthol and icilin. Related macrolide immunosuppressants act as partial TRPM8 agonists, competing with rapamycin for the same binding site. These findings identify a novel molecular target for rapamycin and provide new insights into the mechanisms of TRPM8 activation, which may assist in the development of therapies targeting this ion channel. Moreover, our findings also indicate that caution is needed when using molecular approaches based on rapamycin-induced dimerization to study ion channel regulation.

neuroscience↗