Assembly and Regulation of the Cytoskeleton
Our primary research interest is the assembly and regulation of the mammalian cytoskeleton. We study the function of cytoskeletal and signaling proteins in diseases that afflict blood cells, including malaria, thrombosis, and cancer.
Host Parasite Interactions in Malaria
The aim of this project is to ascertain the mechanisms of malaria pathogenesis in red blood cells (RBCs) / erythrocytes, with the goal of developing novel therapeutics and vaccines. Because of emerging drug resistance and the lack of an effective vaccine, there is an urgent need to identify new drugs and vaccine targets against malaria. The identification of the malaria parasite invasion proteins (ligands) and their cognate host proteins (receptors) is considered essential for the development of an effective multi-subunit vaccine. In Plasmodium falciparum, the most lethal human malaria parasite, RBC invasion takes place via two distinct molecular pathways involving either a sialic acid-dependent or sialic acid-independent mechanism. Using a multidisciplinary approach, we identified an essential role of host RBC membrane complex containing Band 3 (anion exchanger-1) and Glycophorin A (GPA) during the parasite invasion of RBCs (Fig. 1). Our model provides a rationale for the exclusive selectivity of the merozoite attachment to RBCs in circulation since the Band 3-GPA complex is not expressed on other blood cells. The malaria parasite exploits surface MSP1 to co-anchor with Band 3 and GPA as host receptor and co-receptor complex in order to enter red blood cells. Currently, we are employing phage display screens to identify additional components of the parasite invasion complex to be included in the blood-stage malaria vaccine.
Figure 1. Erythrocyte/RBC invasion by the malaria parasite
A distinctive feature of Plasmodium falciparum biology is sequestration, the adhesion of infected RBCs to host endothelial cells lining the blood vessels. Sequestration occurs in major organs including the heart, lung, kidney, liver, and brain. Human cerebral malaria has been strongly correlated with excessive sequestration of infected RBCs resulting in decreased microcirculatory flow and reduced oxygen supply to the brain. Infected RBCs display surface bumps known as knobs that mediate the attachment of infected cells to blood vessels (Fig. 2). Two key malaria proteins regulate the attachment of knobs to endothelial cells. The first protein designated as PfEMP-1 / VAR is synthesized by the intracellular parasite and inserted as a receptor in the RBC membrane knobs. The second protein is a histidine-rich molecule designated as KAHRP. It is also synthesized by the intracellular parasite and deposited on the cytoplasmic surface of infected RBC membrane knobs. We are investigating the mechanism of the PfEMP1-KAHRP interactions to develop novel therapeutics against cerebral malaria.
Figure 2. Visualization of knobs in human erythrocytes.
Calpains and G Protein Signaling
Calcium is a fundamental secondary messenger required for diverse signaling pathways. Calpain-1, a calcium-dependent cysteine protease, regulates multiple signaling and cytoskeletal functions in platelets, erythrocytes, neurons, and many other cells. We generated the first mouse model of calpain-1 deficiency. Using a double knockout mouse model, we identified PTP1B, a major protein tyrosine phosphatase, as a physiological substrate of calpain-1 in platelets. We are also investigating the function of calpain-1 substrates such as dematin and adducin in cell secretion, adhesion, motility, and apoptosis pathways. We use genetically modified mice lacking dematin and adducin as models to facilitate these studies. It is known that calpain protease releases the FERM domain of Talin, thus regulating integrin activation. In collaboration with Guy Le Breton, we discovered a novel calcium-regulated Gα13 switch region 2 signaling mechanism for integrin activation via Talin (Fig. 3). These studies are potentially relevant to the development of new cardiovascular and cancer therapeutics.
Figure 3. A model of integrin activation by Gα13 and Talin complex in human platelets.
Scaffolding Proteins and Kinesins
Cell polarity is a fundamental process required for the function of many cell types. Loss of cell polarity leads to developmental abnormalities and cancer. We are investigating the function of membrane-associated guanylate kinase homologues (MAGUKs) in the regulation of cell polarity signaling pathways via kinesin motors. MAGUKs are scaffolding proteins composed of PDZ, SH3, and GUK domains regulating multiple protein-protein interactions in erythrocytes, lymphocytes, neutrophils, neurons, and epithelia (Fig. 4). My lab provided the first evidence identifying a cytoskeletal-binding interface in MAGUKs via Protein 4.1. These studies led to us naming and characterizing the FERM domain, now a widely recognized structural module involved in multiple signaling and scaffolding proteins. While investigating the role of MAGUKs in cell polarity pathways, we discovered a new mechanism for the intracellular transport of PIP3, a product of phosphatidylinositol 3-kinase, via GAKIN, a kinesin-3 family motor protein. GAKIN, also called KIF13B, was first identified and cloned in our laboratory. Using MPP1, KIF13B, and KIF13A mouse models, developed in our laboratory, we are currently investigating the mechanisms of intracellular cholesterol transport and lipid homeostasis.
Figure 4. Domain organization of selected MAGUKs