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1.  Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis 
G-protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G-protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and GTPase-accelerating protein (GAP), EhRGS-RhoGEF, in a nucleotide state-selective fashion. Co-expression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the DH domain Rho-binding surface and the PH domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical RGS domain rather than a RhoGEF-RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G-protein cross-talk.
PMCID: PMC3545058  PMID: 23260656
2.  Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica 
PLoS Pathogens  2012;8(11):e1003040.
Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.
Author Summary
Entamoeba histolytica causes an estimated 50 million intestinal infections and 100,000 deaths per year worldwide. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, constituting a signaling pathway which, when perturbed, is seen to regulate multiple cellular processes required for pathogenesis. Like mammalian counterparts, EhGα1 forms a heterotrimer with EhGβγ that is dependent on guanine nucleotide exchange and hydrolysis. Despite engaging a classical G-protein effector, EhRGS-RhoGEF, EhGα1 diverges from mammalian Gα subunits and cannot be classified within mammalian Gα subfamilies, as highlighted by distinct structural features in our crystal structure of EhGα1 in the inactive conformation. To identify roles of G-protein signaling in pathogenesis-related cellular processes, we engineered trophozoites for inducible expression of EhGα1 or a dominant negative mutant, finding that G-protein signaling perturbation affects host cell attachment and the related process of contact-dependent killing, as well as trophozoite migration and Matrigel transmigration. A transcriptomic comparison of our engineered strains revealed differential expression of known virulence-associated genes, including amoebapores and cytotoxic cysteine proteases. The expression data suggested, and biochemical experiments confirmed, that cysteine protease secretion is altered upon G-protein overexpression, identifying a mechanism by which pathogenesis-related trophozoite behaviors are perturbed. In summary, E. histolytica encodes a vital heterotrimeric G-protein signaling pathway that is likely amenable to pharmacologic manipulation.
PMCID: PMC3499586  PMID: 23166501
3.  A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization 
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg2+ concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
PMCID: PMC2957273  PMID: 20662737
4.  A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance 
i. Summary
Surface Plasmon Resonance (SPR) is a robust method to detect and quantify macromolecular interactions; however, to measure binding interactions, one component must be immobilized on a sensor surface. This is typically achieved using covalent immobilization via free amines or thiols, or noncovalent immobilization using high affinity interactions such as biotin/streptavidin or antibody/antigen. In this Chapter we describe a robust method to covalently immobilize His6 fusion proteins on the sensor surface for SPR analysis.
PMCID: PMC3031178  PMID: 20217615
Biacore; SPR; Capture Coupling; Immobilization; Hexahistidine; His-tag
5.  A High-Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction 
The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high-throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z′-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z′-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
PMCID: PMC2440576  PMID: 18537560
Fluorescence anisotropy; fluorescence polarization; GoLoco motif; heterotrimeric G-proteins; high-throughput screening

Results 1-5 (5)