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 is essential to control the levels of cellular regulatory proteins and is a critical element of protein quality control systems. Protein degradation is performed by multimeric ATP-dependent proteases, which have three constituents: a substrate recognition domain, an ATP-driven protein unfoldase, and a tightly associated self-compartmentalized protease. Our research encompasses structural and biochemical studies of the ATP-dependent Clp proteases from bacteria and human mitochondria as well as analysis of their biological activities and functions. Current efforts focus on four major areas: the role of ClpAP and ClpS in degradation of proteins containing N-degrons;structural dynamics of ClpP and the mechanism by which unfolded proteins enter the degradation chamber;substrate recognition by human mitochondrial ClpX and the role of ClpXP in mitochondrial function and cell growth;and the role of ClpP in modulating the sensitivity of human cells to anti-cancer drugs such as cisplatin.The N-end rule is a universal mechanism by which proteins are targeted for degradation by virtue of a subset of N-terminal amino acids, referred to as N-degrons. N-degrons are recognized by components of the degradative machinery (N-recognins) enabling the proteins to be targeted for degradation by ATP-dependent proteases. In E. coli, ClpS binds N-degrons and delivers substrates to the ClpAP complex. The N-degrons in E. coli are leucine, phenylalanine, tyrosine, and tryptophan. Proteins with N-terminal lysine and arginine acquire an N-degron through the action of Aat, an amino transferase that adds leucine or phenylalanine to either of these N-termini. Because newly synthesized proteins do not as a rule contain these N-terminal amino acids, N-degrons must arise as a result of post-translational modification or through errors in protein translation. We used a ClpS affinity column to capture proteins with N-degrons and recovered >60 unique proteins. Many of the proteins were seen only in cells with a functional Phe/Leu-aminotransferase, indicating that they first arise with an N-terminal lysine or arginine, which is subsequently modified by Aat. We assayed several mutants with defective protein initiation factors, but none has shown an increase in the number or yield of N-degrons. Many proteins with N-degrons were truncated versions of full-length proteins and appear to result from partial proteolysis. We analyzed more than 50 E. coli mutants lacking single peptidases and found that several proteins with N-degrons did not appear in cells lacking specific peptidases. We are continuing the analysis with cells lacking multiple peptidases to establish links between specific sets of peptidases and the appearance of specific proteins with N-degrons. Our hypothesis is that proteins are subject to constant surveillance to assess their functionality as reflected by their folding integrity or their association with functional partners. Studies with ClpP are focused on the mechanism of cell death that results from activation by the acyldepsipeptide antibiotic, ADEP, and the structural changes that are needed to allow substrate entry into the degradation chamber. ADEP binding to ClpP allows it to target nascent polypeptides and unstructured regions of functional proteins. We hypothesize that cell death results from destruction of one or a few critical cellular proteins either before they are fully synthesized or when their unstructured regions are accessible as a result of changes in interactions with ligands or interacting partners. We are trapping proteins in vivo using a mutant of ClpP that lacks proteolytic activity and will identify the trapped proteins using mass spectrometry to determine the global extent of protein degradation by ClpP in the presence of ADEP.ClpP can exist in two states, one with the handle regions interlaced to expand the degradation chamber and allow proteolysis and another with the handles in a collapsed state, which is either a latent state or is a transient intermediate formed during the degradation cycle to allow release of peptide products. We found that Zn inhibits ClpP, possibly by stabilizing the collapsed state. We are growing crystals of ClpP in the presence of Zn to obtain structural data that will provide insight about the dynamics of ring-ring interactions and their affect on activity. Recently, we began collaboration with the laboratory of Alfred Goldberg at Harvard Medical School, who has provided us with purified ClpP from Mycobacterium tuberculosis. M. tuberculosis has two isozymes of ClpP, which interact with one another to form a mixed tetradecamer needed to express enzymatic activity. The presence of two forms of ClpP in one complex will facilitate structural analysis of the ring interactions, for example, by allowing assembly of tetradecamers in which only one ring is mutated. We have found conditions that give diffractable crystals of Mbt-ClpP and expect to have a structure of the native protein this year. M. tuberculosis ClpP is a promising target, because it is essential for growth. A crystal structure should guide the design of small molecules inhibitors that might serve as leads for the development of compounds with therapeutic potential.The goal of our studies of human ClpX and ClpP is to define their functions in mitochondria and to discover why they are needed for mitochondrial integrity and cell survival. Depletion of hClpP or hClpX following treatment with siRNA leads to cell death. We have analyzed the changes in the mitochondrial proteome in response to changes in hClpP. More than 30 proteins are increased within 16 hours of depletion of hClpP, many of which are involved in stress responses. ADEP induces cellular stress and kills human cells. Over expression of wild type but not inactive mutants of ClpP renders cells more sensitive to ADEP. Proteomics analysis of cells after exposure to ADEP revealed many proteins associated with stress responses that were elevated. Levels of a major anion transporter were also altered after ADEP treatment. The role ClpP plays in mitochondrial anion flux is under further study.Down regulation of hClpP sensitizes cells to cisplatin. Remarkably, cells independently selected for resistance to cisplatin have been found to have elevated levels of expression of ClpX and ClpP. We found that the levels of ClpP affect accumulation of cisplatin in several human cell lines. Total cisplatin accumulation increases when ClpP is knocked down, and there is a marked increase in cisplatin-mediated damage to mitochondrial DNA. Our results suggest that damage to mitochondrial DNA is important in inducing apoptosis following cisplatin treatment. To investigate the link between ClpP and cisplatin accumulation, we measured the levels of copper transporters when ClpP levels were manipulated. Cisplatin is believed to hitchhike on the copper transporters to enter and exit cells. No changes were observed in the major copper transporter, Ctr1, when ClpP was over-expressed or knocked down. However, there was a significant correlation between the levels of ClpP and the levels of the copper efflux pump, ATP7A. ATP7A decreased in proportion to the decrease in hClpP and increased when hClpP was over expressed. The data point to an indirect role for hClpP in affecting ATP7A. We hypothesize that hClpP affects metal ion flux between the mitochondria and the cytosol, which in turn leads to up or down regulation of ATP7A and renders the cell more or less sensitive to cisplatin. We are conducting whole cell assays to measure mitochondrial ion flux, mitochondrial membrane potential, and other mitochondrial activities following knock down of hClpP.

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