Research in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of cellular regulatory proteins and is an essential element of protein quality control systems. Protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multimeric complexes made up of three essential components: a recognition domain that interacts with specific signals in target proteins, an ATP-driven protein unfoldase that structurally disrupts the bound protein and translocates it to the third component, and a tightly associated self-compartmentalized protease. Our research encompasses structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from bacteria and from human mitochondria and assays of their biological activities and the metabolic and regulatory pathways in which they function. Studies are focused on four major areas: the basis for substrate selection by ClpA, ClpX, and Lon;structural dynamics of ClpP and the mechanism by which unfolded proteins enter the degradation chamber;conformational changes in the AAA+ domains of Clps and Lon that contribute to their activities;and the role of human ClpXP in mitochondrial function and signaling under conditions of cellular stress. One universal mechanism of protein recognition operates by controlled exposure of a subset of amino acids at the N-terminus of proteins (N-degrons). Binding of N-degrons by components of the degradative machinery (N-recognins) allows these proteins to be targeted for degradation by ATP-dependent proteases. In bacterial cells ClpAP degrades proteins with N-degrons, and the adaptor protein, ClpS, is the N-recognin. We found that delivery of substrates to the ClpAP complex occurs with only one molecule of ClpS per ClpA hexamer despite the presence of 6 equivalent N-domains capable of binding ClpS. Allowing only one substrate at a time to enter the axial channels of ClpA avoids steric clashes and more efficient protein translocation. ClpS has a bipartite binding mode in which the globular domain interacts with a ClpA N-domain and the N-terminal 20 amino acids interact with the axial channel. The ClpS N-terminal region plays a role in limiting the binding stoichiometry and also in facilitating hand-off of substrates to ClpA. Structural studies of the ternary complex of ClpS and protein substrate bound to ClpA hexamers using cryo electron microscopy and x-ray crystallography will provide insight as to how the ClpS N-terminus binds to ClpA and how the binding changes upon ATP-driven substrate transfer. Related studies are designed to learn how proteins with N-degrons arise and to identify all the proteins that acquire N-degrons in E. coli. We have used a ClpS affinity column to capture proteins with exposed N-degrons and have identified >50 unique proteins bearing N-degrons. We found that the number of proteins isolated depends on how much ClpS and ClpA are present in the cells confirming that the proteins are substrates for degradation by ClpAP/ClpS. We have also found some proteins acquire N-degrons in a process dependent on the enzyme, Phe-aminotransferase, which modifies the N-terminus of proteins to make them easily recognized by ClpS. One hypothesis we are testing is that errors in translation initiation evoke the activity of a system for tagging the N-terminus of the protein, thereby targeting it for degradation. We are examining strains defective in translation initiation and are subjecting cultures to nutrient limitation to cause increased errors in translation and are examining the number and nature of the proteins that acquire novel N-degrons. We found that when cells carry a mutation in aat, the gene for the aminotransferase, they grow very poorly when one of the translation initiation factors required for fidelity is also mutated. This finding suggests a link between the activity of the aminotransferase and fidelity of translation. Studies with ClpP have been focused on the structural changes that are needed to allow substrate entry into the degradation chamber. Cryo electron microscopy shows that the axial pore of ClpP expands to a diameter of greater than 18 angstroms when ClpA binds. With our collaborators, we obtained the crystal structure of ClpP in the open state induced when the acyldepsipeptide antibiotic, ADEP1, is bound. The open-channel form of ClpP can take up unfolded proteins and is highly activated for peptide degradation. When ADEP is added to cells it binds to ClpP and allows it to target nascent polypeptides during protein synthesis, and degradation of the newly synthesized proteins before they can fold leads to cell death. We hypothesize that cell death results from destruction of one or a few critical cellular proteins before they can be fully synthesized rather then a global drop in protein synthesis. We are trapping nascent proteins in vivo using a mutant form of ClpP, and we will identify the trapped proteins using mass spectrometry and then use genetic methods to define the ones that must be constantly synthesized to allow cells to grow. We have begun to search for other compounds that can bind to ClpP and induce the open state of ClpP. These compounds will be lead molecules for the development of potential novel antibiotics and will serve as additional ligands for in vitro studies aimed at understanding the mechanism by which ClpP undergoes the structural changes that open its substrate access channel. The major goal of our studies of human ClpX and ClpP is to define their functions within mitochondria and to discover why they are needed for mitochondrial integrity and for cell survival. Depletion of hClpP or hClpX following treatment with siRNA leads to cell death in part at least to induction of apoptosis. Down regulation of hClpP sensitizes cells to apoptotic cell death in response to DNA damage, death receptor binding, and kinase inhibition. The similarity in response to 3 divergent stress-signaling pathways suggests that hClpP alters the basal structure or physiology of mitochondria. Down regulation of hClpP sensitizes cells to various drugs that have been used to treat cancer and partially reverse the drug resistance of multidrug resistant cells. We have found that cells selected for resistance to cisplatin have elevated levels of expression of ClpX and ClpP. Out current efforts are focused on describing the changes in the mitochondrial proteome in response to depletion and over expression of hClpP and hClpX. Analysis of 2D gels followed by mass spectrometry has identified more than 30 proteins whose levels increased within 16 hours of depletion of hClpP, many of which are involved in the stress responses. We have also examined the effects of derivatives of the antibiotic ascochlorin that induce endoplasmic reticulum stress on the levels of mitochondrial ClpP and Lon proteases. One derivative, AS-6, leads to ER stress, induces autophagy and leads to apoptotic cell death. Interestingly, ClpP levels transiently increase and then decline after AS-6 treatment, and we are investigating whether the loss of ClpP is responsible for induction of apoptosis under these conditions. We have found that ADEP, which also activates human ClpP in vitro, induces cellular stress and kills cells. Over expression of wild type but not inactive mutants of ClpP renders cells more sensitive to ADEP. We are in the process of isolating potential targets of ADEP-activated ClpP by trapping them using an inactive ClpP mutant, pulling down the trapped substrate/ClpP complex, and identifying the trapped proteins by digestion followed by tandem MS/MS.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Zhang, Yang; Maurizi, Michael R (2016) Mitochondrial ClpP activity is required for cisplatin resistance in human cells. Biochim Biophys Acta 1862:252-64
Maurizi, Michael R; Stan, George (2013) ClpX shifts into high gear to unfold stable proteins. Cell 155:502-4
Humbard, Matthew A; Surkov, Serhiy; De Donatis, Gian Marco et al. (2013) The N-degradome of Escherichia coli: LIMITED PROTEOLYSIS IN VIVO GENERATES A LARGE POOL OF PROTEINS BEARING N-DEGRONS. J Biol Chem 288:28913-24
Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory et al. (2012) The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features. Plant Mol Biol 80:189-202
Kang, Jeong Han; Chang, Young-Chae; Maurizi, Michael R (2012) 4-O-carboxymethyl ascochlorin causes ER stress and induced autophagy in human hepatocellular carcinoma cells. J Biol Chem 287:15661-71
Effantin, Grégory; Ishikawa, Takashi; De Donatis, Gian Marco et al. (2010) Local and global mobility in the ClpA AAA+ chaperone detected by cryo-electron microscopy: functional connotations. Structure 18:553-62
Li, Mi; Gustchina, Alla; Rasulova, Fatima S et al. (2010) Structure of the N-terminal fragment of Escherichia coli Lon protease. Acta Crystallogr D Biol Crystallogr 66:865-73
Effantin, Gregory; Maurizi, Michael R; Steven, Alasdair C (2010) Binding of the ClpA unfoldase opens the axial gate of ClpP peptidase. J Biol Chem 285:14834-40
De Donatis, Gian Marco; Singh, Satyendra K; Viswanathan, Sarada et al. (2010) A single ClpS monomer is sufficient to direct the activity of the ClpA hexamer. J Biol Chem 285:8771-81