Additional details are provided in em SI Experimental Procedures /em
Additional details are provided in em SI Experimental Procedures /em . Cell Viability. detect 2M in human blood plasma after native polyacrylamide gel electrophoresis. In = 3; SD). *Significant increase in ANS-associated fluorescence of NaOCl-treated 2M compared with the native control (Student test; 0.01). All fluorescence measurements are shown in arbitrary fluorescence units (AFU). By Western blot analysis, 2M is predominantly detected in its native tetrameric form in human blood plasma (Fig. 1and and and and and and and Although it is conceivable that, in the presence of NaOCl, 2M may become covalently linked to bFGN or bLDL, this possibility is excluded by the observation that, after heating and reduction, the bound 2M migrates at positions corresponding to its dimeric form (360 kDa; which is the result of oxidative intersubunit cross-linking) (27) or its monomeric form (180 kDa) or as heat-liberated fragments (41), and other species are not detected. Furthermore, relative to native 2M, preoxidized 2M preferentially associates with preoxidized Rabbit Polyclonal to SFRS11 bFGN and preoxidized bLDL, indicating that the oxidized proteins bind through noncovalent interactions (Fig. 3and show results of flow cytometric analyses of the binding of native and ox bA1C42 to RAW 264.7 macrophages. The cells were either preincubated with RAP, which inhibits lipoprotein receptors, or untreated before incubation with bA1C42 (2.5 M in HBB) at 4 C. The LR-D binding and LR-I binding were determined as described in = 3 SD) in arbitrary fluorescence units (AFUs) and adjusted for background fluorescence. (and then preincubated for 20 min at RT native or ox 2M at an approximate molar ratio of 2M:bA1C42 of 1 1:10. (= 6; SD) absorbance at 595 nm (A595 nm). ^Significant increases in cell surface bA1C42 binding and vsignificant decreases in cell surface bA1C42 binding (Student test; 0.05 in both cases). *Significantly increased cell surface binding of ox bA1C42 compared to bA1C42 (Student test; 0.01). **Significantly increased cell viability compared with cells treated with A1C42 alone (Student test; 0.01). Preincubation of bA1C42 with native 2M significantly reduces the subsequent binding of bA1C42 to RAW 264.7 cells (Fig. 6and and and 2 and em F /em ). Similarly, hypochlorite-modified 2M tetramers and dimers both show increased binding to IL-2 and IL-6 (29), and PT2977 treatment of 2M with detergent produces tetramers and dimers that bind more strongly to the amyloidogenic protein 2-microglobulin than does native 2M (16). Partial opening of the dimer interface to expose the relevant binding site(s) on tetrameric 2M or the formation of 2M tetramers from modified, dissociated dimers may explain our results and the results of the aforementioned studies, although it is also possible that other structural changes are responsible. We have further shown that 2M dimers generated by treatment with NaCSN also have enhanced chaperone activity (Fig. S2), indicating that physical dissociation from a tetramer to a dimer is sufficient to increase chaperone activity, independent of any requirement for oxidation. Notably, it has been shown that 2M dimers generated by treatment with NaCSN also bind to LRP (35). PT2977 Therefore, if the dissociation of native 2M into dimers is sufficient to enhance its chaperone activity, its binding to cytokines, and its clearance through LRP, it may be possible to exploit this effect in the development of novel therapeutics. Role of 2M in Inflammation. Inflammation is a state in which many stresses capable of inducing protein misfolding and aggregation are elevated; therefore, it is likely that proteostasis mechanisms will be enhanced during inflammatory events. Moreover, it has been shown that many acute-phase proteins are susceptible to stress-induced misfolding and major PT2977 endogenous clients for holdase chaperones in human blood plasma (65C67). To survive periods of increased physiological stress, cells use several different strategies to prevent the accumulation of misfolded proteins (68C71). In this study, we show that hypochlorite-induced structural modifications enhance the ability of 2M to inhibit the amorphous aggregation of globular proteins and the fibrillar aggregation of A1C42. After their binding to oxidized 2M, misfolded client proteins are directed to macrophage lipoprotein receptors, which are a potential route for their clearance.