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Ladokhin Lab

photo of ladokhin lab members in 2014

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 >>

Ladokhin Lab Photo Archive


Major Research Interests

I. Apoptotic regulation by the Bcl-2 protein family

Figure 1 - diagram of functional interactions between Bcl-2 family proteins
Figure 1. Many functional interactions between Bcl-2 family proteins that regulate apoptosis occur only in membranes. The mechanisms by which the membrane induces conformational changes and modulates proteinprotein interactions remain largely unknown.

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

Figure 2 - digital images captured on microscope of cells
Figure 2. (Left) Evidence for the novel mechanism of pHLIP targeting by Ca2+. pH- vs Ca2+-dependent insertion of dye-labeled pHLIP into MDA-MB-231 cancer cells (images). Presence of 2 mM Ca2+ improves cellular delivery of MMAF drug, conjugated to C-terminus of pHLIP, and reduces the viability of cultured HeLa cells (graph). (Right) Biophysical studies in support of our model. Vesicle insertion of pHLIP detected by Trp fluorescence (graph) shows that additions of divalent cations result in similar spectral shift as acidification, suggesting pHLIP insertion to occur at pH as high as 10. A schematic representation of the proposed interactions of divalent cations with lipid headgroups and pHLIP anionic sidechains.

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) 


III. Retargeting bacterial toxins to tumors

Figure 3 - revised scheme of membrane interactions of cancer-targeting pHLIP peptides diagram
Figure 3. Diphtheria toxin’s translocation (T) domain plays a central role in cellular entry, occurring via the endosomal pathway. Its function is to translocate the catalytic domain across the membrane in response to acidification of the endosomal interior. Our previous studies reveled that T-domain undergoes a series of conformational changes schematically depicted on the graph. This refolding/insertion pathway ultimately results in the pH-dependent translocation of the N-terminus-attached catalytic domain across the membrane. We propose to take advantage of this process and retarget the T-domain to the acidic environment created by tumors, thus ensuring selective delivery of anticancer therapies.

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).


IV. Biophysical methods: Fluorescence Spectroscopy (FCS, FRET, lifetime spectroscopy, depth-dependent fluorescence quenching in membranes); Molecular Dynamics (MD) computer simulations; Thermodynamic analysis.   


KU School of Medicine

University of Kansas Medical Center
Biochemistry and Molecular Biology
3901 Rainbow Boulevard
1080 HLSIC ,  Mailstop 3030
Kansas City, KS 66160-7421