One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.

Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function.

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.