Biochemistry and Pharmacology - Research Publications
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ItemSize analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge.(Springer Nature, 2013)Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.
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ItemPulse shape analysis (PulSA) to track protein translocalization in cells by flow cytometry: applications for polyglutamine aggregation.(Springer Nature, 2013)Pulse shape analysis (PulSA) is a flow cytometry-based method that can be used to study protein localization patterns in cells. Examples for its use include tracking the formation of inclusion bodies of polyglutamine-expanded proteins and other aggregating proteins. The method can also be used for phenomena relating to protein movements in cells such as translocation from the cytoplasm to the nucleus, trafficking from the plasma membrane to the Golgi, and stress granule formation. An attractive feature is its capacity to quantify these parameters in whole-cell populations very quickly and in high throughput. We describe the basic experimental details for performing PulSA using expression of GFP-tagged proteins, endogenous proteins labelled immunofluorescently, and organelle dyes.
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ItemNo Preview AvailableThe Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013-03-22)Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.
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ItemSimultaneous Binding of the Anti-Cancer IgM Monoclonal Antibody PAT-SM6 to Low Density Lipoproteins and GRP78(PUBLIC LIBRARY SCIENCE, 2013-04-19)The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface.
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ItemA Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013-12-27)Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell.
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ItemTyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src(PUBLIC LIBRARY SCIENCE, 2013-07-26)c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.