Actin – numerous functions of one protein
Actin is one of the most abundant and functionally versatile proteins on our planet. As a major component of the cytoskeleton, actin serves as a track for myosin-based motility and thus is involved in muscle contraction, organelle transport, and closure of the contractile ring. In addition, polymerization of actin itself also serves motor purposes and thus drives exo- and endocytosis, cell division, migration, invasion, and other cellular processes of high physiological and pathological relevance. Thus invasion of cancer cells underlies metastatic dissemination of human cancers and is actively pursued as a potential therapeutic target. Currently, our interests in this area are focused on the directions outlined below.
Actin is involved in the pathogenesis of infectious diseases as an element of the innate immune system, but also as a target that can be hijacked by many bacterial and viral toxins. We are interested in deciphering the role of actin in both phenomena.
- Thus, we investigate properties and the role of actin binding protein L-plastin in phagocytosis and migration of immune cells. There are three plastin isoforms in humans all of which seem to bind and bundle actin filaments in a Ca(2+)-dependent manner. Interestingly, expression of L-plastin, but not the other two isoforms is frequently elevated in tumors and associated with their increased metastatic activity. We compare properties of L-plastin with two other human plastins (T- and I-plastins) in order to understand what makes the L-isoform particularly suitable for invasion.
- Next, we investigate the molecular and cellular mechanisms of the ACD (actin crosslinking domain) toxin from Vibrio cholerae and several other Gram-negative bacteria. ACD covalently cross-links actin subunits into polymerization-defective oligomers and thus hampers the ability of immune cells to engulf and eliminate bacteria. Our data suggest that ACD is highly effective even at very low doses, when only a small population of actin in the affected cells is cross-linked. We propose that the cross-linked oligomers interfere with the activity of several vital cytoskeletal proteins (e.g. nucleators of actin polymerization formins) when present at very low concentrations leading to i) impaired ability of macrophages to fight bacterial cells and to ii) increased permeability of intestinal epithelial layers.
- Lately, the role of actin in numerous nuclear functions has been proposed, but progress in understanding of these roles at the cellular level is hampered by the lack of tools for selective targeting nuclear but not the cytoplasmic actin. Our lab is actively involved in development of such tools by utilizing actin-specific activities of bacterial toxins (ACD, SipA, etc.) and mammalian actin modifiers.
Selective targeting and elimination of cancer cells with bacterial toxins
- Several bacterial toxins have attracted significant interest as potent antitumor agents due to their selectivity, ability to penetrate the cell membrane, high resistance against host defense systems, and demonstrated capacity to kill cells by mechanisms independent of the drug-resistant phenotypes of most tumors. Yet, the specificity of the currently recognized toxins towards tumor cells is limited due to a broad cross-specificity of the toxin receptors. We propose to deliver split variants of potent bacterial toxins via two different pathways uniquely represented only on the surface of cancer cells. The idea is that the fully functional toxin will be assembled only in the cytoplasm of a doubly targeted cancer cell.
Selective inactivation of bacterial toxins by human defensins
- Defensins are a family of short cationic immune peptides with a broad repertoire of anti-microbial activities. Defensins disorganize bacterial cell membranes and inactivate protein bacterial toxins while showing little effect on host’s proteins. Yet, the amazing selectivity of defensins against various unrelated toxins is not well understood, nor has a coherent unifying hypothesis been proposed to explain this selectivity. We have developed a keen interest in deciphering the mechanisms of amazingly selective inactivation of bacterial toxins by human defensins. We propose that many bacterial toxins share an elusive common property that can be efficiently exploited by human defensins and we are currently in the process of identification of these elusive properties. The data acquired in the course of the project should lead to the development of rationally designed anti-toxin therapeutic agents.
To accomplish our goals we employ highly interdisciplinary combination of biochemical, biophysical, and cell biology approaches, including but not limited to methods of fluorescence spectroscopy, circular dichroism, calorimetry, mass spectrometry, electron microscopy, cell imaging, fluorescent microscopy, and others. In addition, we collaborate with several groups for X-ray crystallography, NMR, and computational biology experiments.