Histone acetyltransferase cofactor Trrap is essential for maintaining the hematopoietic stem/progenitor cell pool.

The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.

Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU.

Pseudomonas aeruginosa is an opportunistic pathogen that uses a type III secretion system and four effector proteins to avoid innate immune responses. ExoS, ExoT, ExoY, and ExoU all possess enzymatic activities that disrupt host cellular physiology and prevent bacterial clearance by host defense mechanisms. The specificity of these toxins for eukaryotic cells depends on the presence of substrate targets and eukaryotic cofactors responsible for effector activation. We used a combined biochemical and proteomic approach to identify Cu(2+), Zn(2+)-superoxide dismutase (SOD1) as a cofactor that activates the phospholipase activity of ExoU. Recombinant ExoU (rExoU) was activated in a dose-dependent manner by either bovine liver SOD1 or the yeast ortholog, Sod1p, but not by either Fe or Mn-containing SODs from E. coli or small molecule SOD mimetics. Inhibitor studies indicated that SOD enzymatic activity was not required for the activation of rExoU. The physical interaction between rExoU and SOD was demonstrated by capture techniques using either of the two proteins immobilized onto the solid phase. Identification of SOD as a cofactor allowed us to develop a new assay using a fluorescent substrate to measure the phospholipase activity of rExoU. The ability of SOD to act as a cytoplasmic cofactor stimulating ExoU phospholipase activity has significant implications for the biological activity of the toxin. Further elucidation of the structural mechanism of ExoU activation by this eukaryotic cofactor may provide a rational approach to the design of inhibitors that can diminish tissue damage during infection by ExoU-producing strains of P. aeruginosa.

Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit.

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

Interaction sites between catalytic and regulatory subunits in human protein kinase CK2 holoenzymes as indicated by chemical cross-linking and immunological investigations.

Protein kinase CK2, a heterotetramer composed of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta), has been examined for intermolecular contact sites by methods that allow investigation of the native, unaltered proteins. Antibodies were raised against a series of 11 subunit peptides, affinity purified, and ensured for site specific binding by peptide competition. Chemical cross-linking of CK2 subunits with a hydrophilic carbodiimide and analysis of fused subunits and of CNBr-digested fusion products by immunoblotting with the sequence specific antibodies identified a tight interaction between positions beta55-70 and alpha65-80 (alpha'66-81) of subunits beta and alpha (alpha'), respectively. This was corroborated by cross-linking of subunits with peptides alpha65-80 and beta55-70 by a peptide-based enzyme-linked immunosorbent assay in which peptides bound to wells via C-10 spacer arms are probed for complexing individual subunits and immunoprecipitation with antibodies anti-alpha65-80 and anti-beta55-70, resulting in precipitation but not coprecipitation of subunits. This alpha-beta (alpha'-beta) interaction site obviously is also of functional importance since subunits with attached antibodies cannot reconstitute to the fully active holoenzyme. Indeed, sites beta55-70 and alpha65-80 (alpha'66-81) correspond to an acidic (beta) and a basic (alpha or alpha') domain involved in activity and stability control and in substrate and cosubstrate binding (kinase domain II/III), respectively. By contrast, a number of suspected contact sites were found to be rather loose and not essential for enzyme control as concluded from precipitation behavior of respective antibodies and the toleration of attached antibodies when active holoenzymes were being constituted. At subunit beta, these include the terminal positions beta2-14 and beta204-213, the positions beta97-105 and beta140-156, and, surprisingly, also beta171-186 which have been shown by deletion mutation and peptide replacement studies to represent a positively affecting interaction site. At subunits alpha and alpha', these are the C-terminal positions alpha329 -343 and alpha'336-350. Binding of antibodies to the positions alpha15-27 (alpha'16-28) and position alpha151-166(alpha'152-167), on the other hand, inhibits activity.

Production and application of monoclonal antibodies specific to pyrroloquinoline quinone.

We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that are specific to pyrroloquinoline quinone (PQQ). PQQ-conjugated hemocyanin was used for the immunization of mice and the hybridomas were selected using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay. MAbs 2 and 9 were of the IgG1 isotype. Both could recognize free PQQ, the former probably at the o-quinone and the latter at the opposite side of the molecule. They did not bind with trihydroxyphenylalanine, dihydroxyphenylalanine, 1,2,4-trihydroxybenzene, ascorbic acid, riboflavin, or menadione. In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ. Using mAb 2, a competitive enzyme-linked immunosorbent assay was developed, which enabled us to determine 50 nM-1 microM free PQQ. Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs. However, the results was negative, indicating that the prosthetic groups are not PQQ.

Monoclonal antibody recognizes different quinone moieties in enzymes.

We produced monoclonal antibodies against the coenzyme pyrrolequinoline quinone (PQQ). These antibodies were obtained by immunizing mice with PQQ conjugated to a chemically modified polypeptide in order to induce a strong immune response. Among the various antibodies obtained, one was found to bind (besides PQQ and 6-hydroxydopamine conjugated to carrier proteins) several different quinoenzymes, namely lentil seedling and bovine serum diamine oxidases and methylamine dehydrogenase. This antibody was able to inhibit the catalytic activity of these enzymes. Moreover, the monoclonal antibody recognized different proteins of lentil seeds on Western blots. Even the variable fragment of immunoglobulin heavy chains of this monoclonal antibody expressed in Escherichia coli is able to recognize the active site of different quinoenzymes.