Welcome to the Ladokhin Lab!
We are interested in understanding the fundamentals of the structure and functioning of proteins interacting with lipid membranes. View our publications >>
Major Research Interests
I. Apoptotic regulation by the Bcl-2 protein family
Apoptosis is crucial for proper development and function of cell populations in tissues, and its dysregulation impacts many diseases. Hyperactive apoptosis contributes to neurodegeneration and immunodeficiency, while insufficient apoptosis leads to autoimmunity and cancer, and the ability of cancer cells to avoid apoptosis significantly complicates treatment. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP), which releases apoptotic factors into the cytosol that lead to cell death. MOMP is controlled and executed by the numerous proteins of Bcl-2 family, which include three types (Fig. 1): pro-apoptotic pore formers (e.g., Bax), anti-apoptotic pore inhibitors (e.g., Bcl-xL), and BH3-only regulators (e.g., Bid). These proteins directly interact within the mitochondrial outer membrane (MOM) either to promote or prevent protein conformational changes that lead to formation of an oligomeric pore. Our goal is to understand molecular mechanisms of membrane-induced conformational switching in Bcl-2 proteins in regulation of apoptosis.
II. pHLIP and conformational switching on membrane interfaces
The pH-low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of tumors. A traditional explanation for tumor-targeting by pHLIP is its pH-triggered transmembrane insertion. Recently our lab discovered that the presence of 2 mM Ca2+, which mimics extracellular conditions, induces pHLIP insertion without the need for acidic conditions. We have reported how changes in lipid composition and presence of divalent cations affect its interactions with model and cellular membranes (Fig. 2). We propose that tumor targeting by pHLIP is modulated by the changes in lipid composition of cancer cells (e.g., by exposure of phosphatidylserine to the outer leaflet)
- Ca 2+ -dependent interactions between lipids and the tumor-targeting peptide pHLIP
Divalent Cations and Lipid Composition Modulate Membrane Insertion and Cancer-Targeting Action of pHLIP
III. Retargeting bacterial toxins to tumors
Changes in side-chain protonation are among the most prominent physicochemical signals capable of triggering functionally relevant structural rearrangements. For example, pH-dependent conversion of a protein structure from a water-soluble to membrane-inserted form is a key step in many processes, including cellular entry of bacterial toxins, colicins, and viruses. Remarkably, the protonation phenomenon is also central to efforts to target acidic cancer tissues and other maladies. We propose to combine these two aspects of the protonation by retargeting diphtheria toxin translocation (T) domain to deliver drugs into cancer cells via proton-induced conformational switching (Fig. 3).
Conformational switching, refolding and membrane insertion of the diphtheria toxin translocation domain
IV. Biophysical methods: Fluorescence Spectroscopy (FCS, FRET, lifetime spectroscopy, depth-dependent fluorescence quenching in membranes); Molecular Dynamics (MD) computer simulations; Thermodynamic analysis.
- Expanding MPEx Hydropathy Analysis to Account for Electrostatic Contributions to Protein Interactions with Anionic Membranes
- Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age
- How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?