Research conducted in the Biochemistry of Proteins Section is focused on basic mechanisms and regulation of protein degradation in bacterial and human cells. Controlled intracellular protein degradation is vitally important, serving both regulatory and protein quality control functions. Most intracellular protein degradation is carried out by multi-component, self-compartmentalized ATP-dependent proteases, which selectively screen potential targets, control access to sequestered proteolytic sites, and can generate discrete degradation products that are recycled to amino acids or sometimes serve as signaling messengers and activators of various cellular responses. Our efforts have been directed toward characterization of the structural and biochemical properties of the ATP-dependent Clp and Lon proteases. In the last year, progress has been made in several areas. We have (in collaboration with Dr. Bijan Ahvazi, NIAMS) refined and published the crystal structure of human mitochondrial ClpP, which has provided the basis for a unique model for the role of the N-terminal peptide of ClpP in regulating or facilitating the passage of substrates through the translocation channel into the active site chamber. Mutagenesis studies have shown that deletion of more than one amino acid from the N-terminus leads to a dramatic decrease or total loss of ClpP activity. In other studies done in collaboration with Dr. Ann Ginsburg, NHLBI, we have shown that human ClpP forms stable heptamers that require the chaperone component, ClpX, to assemble into the bilayered structure with sequestered active sites. These data suggest that assembly of ClpP may be regulated in human cells as a means of controlling the amount or the specificity of ClpP activity. We have obtained stable human cancer cell lines over-expressing either active or inactive mutant forms of human ClpP. Preliminary data suggests that excess human ClpP has effect on the timing or extent of cisplatin-induced apoptosis. We will examine changes in the level of pro- and anti-apoptotic proteins in mitochondria in response to altered expression of ClpP. Efforts are underway to manipulate the cellular content of human ClpP using siRNA techniques and to determine the role of human ClpX on the cellular responses to ClpP so far observed. Biochemical studies of ClpAP and ClpXP have provided several intriguing insights regarding substrate binding by the chaperone component. Short peptides containing sequence motifs recognized by ClpA or ClpX have been shown to bind with a stoichiometry of one peptide per hexamer. Interesting, peptides with different sequence motifs recognized by ClpA compete for binding, indicating that peptide interaction sites may be deformable and adaptable to different motifs or that the sites are structurally complex and contain multiple docking sites that bind different motifs. Because ClpA is a six-fold symmetric complex, limiting binding to one peptide requires a mechanism to exclude peptides from the remaining equivalent sites following binding of the first ligand. We are investigating whether Clp chaperones undergo a conformational change on peptide binding to explain negative cooperativity of binding or whether the peptide binding sites lie close together near the center of the ring and either overlap or sterically interfere with each other. Studies with the adaptor protein, ClpS, which can alter the substrate preference of ClpA, show that ClpS exerts its effect on ClpA also at a stoichiometry of one ClpS per hexamer.