Measuring G Protein Activation with Spectrally Resolved Fluorescence Fluctuation Spectroscopy

G protein-coupled receptor signaling has been posited to occur through either collision coupling or pre-assembled complexes with G protein transducers. To investigate the dynamics of G protein signaling, we introduce fluorescence covariance matrix analysis (FCMA), a novel implementation of fluorescence cumulant analysis applied to spectrally resolved fluorescence images. We labeled the GPCR, Gα, and Gβγ units with distinct fluorescent protein labels and we applied FCMA to measure directly the complex formation during stimulation of dopamine and adrenergic receptors. To determine the prevalence of hetero-oligomers, we compared the GPCR data to those from control samples expressing three fluorescent protein labels with known stoichiometries. Interactions between Gα and Gβγ subunits determined by FCMA were sensitive to stimulation with GPCR ligands. However, GPCR/G protein interactions were too weak to be distinguished from background. These findings support a collision coupling mechanism rather than pre-assembled complexes for the two GPCRs studied.

Yb to Tb Cooperative Upconversion in Supramolecularly Assembled Complexes in a Solution

The podand-type ligand L, based on a tertiary amine substituted by three pyridyl-6-phosphonic acid functions, forms hydrated complexes with Ln3+ cations. The luminescence properties of the YbL complex were studied in D2O as a function of the pD and temperature. In basic conditions, increases in the luminescence quantum yield and the excited state lifetime of the Yb centered emission associated with the 2F5/2 → 2F7/2 transition were observed and attributed to a change in the hydration number from two water molecules in the first coordination sphere of Yb at acidic pH to a single one in basic conditions. Upon the addition of TbCl3 salts to a solution containing the YbL complex in D2O, heteropolynuclear Yb/Tb species formed, and excitation of the Yb at 980 nm resulted in the observation of the typical visible emission of Tb as a result of a cooperative upconversion (UC) photosensitization process. The UC was further evidenced by the quadratic dependence of the UC emission as a function of the laser power density.

The structures of natively assembled clathrin-coated vesicles

Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

The Structures of Natively Assembled Clathrin Coated Vesicles

Vesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2-appendages crosslink adjacent CHC β-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

Gene silencing in Cryptosporidium: A rapid approach to identify novel targets for drug development

BackgroundCryptosporidiosis is a major cause of diarrheal disease. However, the only drug approved for cryptosporidiosis does not work well in high risk populations. Therefore, novel drugs are urgently needed. Then, the identification of novel is necessary to develop new therapies against this parasite. Recently, we have developed a rapid method to block gene expression in Cryptosporidium by using pre-assembled complexes of Cryptosporidium antisense RNA and human protein with slicer activity (Argonaute 2). We hypothesized that structural proteins, proteases, enzymes nucleotide synthesis and transcription factors are essential for parasite development, thus in this work we knock down expression of 4 selected genes: Actin, Apicomplexan DNA-binding protein (AP2), Rhomboid protein 1 (Rom 1) and nucleoside diphosphate kinase (NDK) and elucidated its role during invasion, proliferation and egress of Cryptosporidium.MethodsWe used protein transfection reagents (PTR) to introduce pre-assembled complexes of antisense RNA and human Argonaute 2 into Cryptosporidium parvum oocysts, the complexes blocked expression of Actin (Act), Transcription factor AP2 (AP2), nucleoside diphosphate kinase (DKN), and rhomboid protein 1 (Rom1). After gene silencing, we evaluated parasite reduction using In vitro models of excystation, invasion, proliferation and egress. We evaluated the potency of ellagic acid, a nucleoside diphosphate kinase inhibitor for anti-cryptosporidial activity using a model of in vitro infection with human HCT-8 cells.ResultsSilencing of Act, AP2, NDK and Rom1 reduce significantly invasion, proliferation and egress of Cryptosporidium. We showed that silencing of NDK markedly inhibited Cryptosporidium proliferation. This was confirmed by demonstration that ellagic acid reduced the number of parasites at micro molar concentrations (EC 50 =15-30 µM) without showing any toxic effect on human cells.ConclusionsOverall the results confirmed the usefulness RNA silencing can be used to identify novel targets for drug development against Cryptosporidium. We identified ellagic acid (EA), a nucleoside diphosphate kinase inhibitor also blocks Cryptosporidium proliferation. Since EA is a dietary supplement approved for human use, then this compound should be studied as a potential treatment for cryptosporidiosis.Author summaryThe World Health Organization reports diarrhea kills around 760,000 children under five every year. Cryptosporidium infection is a leading cause of diarrhea morbidity and mortality. Current therapies to treat this infection are suboptimal, therefore novel treatments are urgently needed. We used genetic tools to identify novel targets for drug development, thus in this work we evaluated the role of 4 genes during Cryptosporidium infection. We demonstrated that silencing of nucleoside-diphosphate kinase (NDK) drastically reduced invasion, proliferation and egress of this parasite. To validate these finding we used the Ellagic acid (EA) an inhibitor of NDK to treat infected intestinal cells. Our results confirmed that the EA blocks parasite proliferation on infected cells. Interestingly we observed that the ellagic acid also has anti cryptosporidial activity by inducing apoptosis. Since EA is a dietary supplement already approved for human use, then this compound has potential to be used as a rapid alternative to treat Cryptosporidiosis.