Small-subunit cysteine-65 substitutions can suppress or induce alterations in the large-subunit catalytic efficiency and holoenzyme thermal stability of ribulose-1,5-bisphosphate carboxylase/oxygenase.

In the green alga Chlamydomonas reinhardtii, an L290F substitution in the chloroplast-encoded large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) causes decreases in carboxylation Vmax, CO2/O2 specificity, and thermal stability. Analysis of photosynthesis-competent revertants selected at the 35 degrees C restrictive temperature identified a rare C65S suppressor substitution in the nuclear-encoded small subunit. C65S enhances catalysis and CO2/O2 specificity in the absence of other wild-type small subunits, and restores thermal stability in vivo. C65S, C65A, and C65P mutant strains were created. C65S and C65A enzymes have normal catalysis, but C65P Rubisco, which contains land-plant Pro, has decreases in carboxylation Vmax/Km and CO2/O2 specificity. In contrast to other small-subunit substitutions that affect specificity, Cys-65 contacts the large subunit, and the C65P substitution does not cause a decrease in holoenzyme thermal stability in vivo or in vitro. Further analysis of the C65P protein may identify structural alterations that influence catalysis separate from those that affect stability.

The dynamic alterations of H2AX complex during DNA repair detected by a proteomic approach reveal the critical roles of Ca(2+)/calmodulin in the ionizing radiation-induced cell cycle arrest.

By using DNA nuclease digestion and a quantitative "dual tagging" proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca(2+)-binding proteins were found in the H2AX complex, the roles of Ca(2+) were examined. The results indicate that Ca(2+)/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

Flightless I homolog negatively modulates the TLR pathway.

To date, much of our knowledge about the signaling networks involved in the innate immune response has come from studies using nonphysiologic model systems rather than actual immune cells. In this study, we used a dual-tagging proteomic strategy to identify the components of the MyD88 signalosome in murine macrophages stimulated with lipid A. This systems approach revealed 16 potential MyD88-interacting partners, one of which, flightless I homolog (Fliih) was verified to interact with MyD88 and was further characterized as a negative regulator of the TLR4-MyD88 pathway. Conversely, a reduction in endogenous Fliih by small-interfering RNA enhanced the activation of NF-kappaB, as well as cytokine production by LPS. Results from immunoprecipitation and a two-hybrid assay further indicated that Fliih directly interfered with the formation of the TLR4-MyD88 signaling complex. These results in turn suggest a new basis for the regulation of the TLR pathway by Fliih.

In vivo dual-tagging proteomic approach in studying signaling pathways in immune response.

Up to date, few successes have been achieved to identify the signaling molecules directly from immune cells due to their low-abundance and dynamic nature. Here, we designed an in vivo dual-tagging quantitative approach that integrated epitope-tagging which allows single affinity purification of the natural complexes formed at real-time, and amino acid-coded mass tagging (AACT) that assists mass spectrometry-based quantitative measurement, to identify the specific components of a signaling complex formed in macrophage cells upon lipopolysaccharide (LPS) stimulation. The sensitivity and accuracy of this quantitative method are significantly higher than those of tandem affinity purification, because the multiple step of purifications are avoided to preserve weakly interacting molecules. We identified a number of proteins that interact with MyD88, a critical adaptor protein in innate immune response, in macrophages upon stimulation. Among those newly identified MyD88-interacting partners, FLAP-1 was found to be an activator of NF-kappaB, the key transcription factor in immune response. This integrated approach provides global information on the functional link between MyD88 and other proteins in transducing the TLR-mediated signal and is generally applicable to in vivo analyses of other signaling pathways.

Assessment of structural and functional divergence far from the large subunit active site of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Despite conservation of three-dimensional structure and active-site residues, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes from divergent species differ with respect to catalytic efficiency and CO2/O2 specificity. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO2 fixation and agricultural productivity. By comparing 500 active-site large subunit sequences from flowering plants with that of the green alga Chlamydomonas reinhardtii, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO2/O2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land plant Val-42 and Ala-53 in the large subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methyl-Cys-256, Lys-258, and Ile-265 to land plant Phe-256, Arg-258, and Val-265 at the bottom of the alpha/beta-barrel active site caused a 10% decrease in CO2/O2 specificity, largely due to an 85% decrease in carboxylation catalytic efficiency (Vmax/Km). Because land plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants. The Rubisco x-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 A away from the active site.