Research in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Protein degradation is essential to control the levels of cellular regulatory proteins and is a critical part of protein quality control systems. Protein degradation is performed by ATP-dependent proteases, which have three constituents: a substrate recognition domain, an ATP-driven protein unfoldase, and an associated self-compartmentalized protease. Our research includes structural and biochemical studies of the Clp proteases from bacteria and human mitochondria and analysis of their biological activities. We have made substantial progress in the past year in understanding intracellular degradation carried out by ClpAP and the adaptor protein, ClpS. The N-end rule is a mechanism by which proteins are targeted for degradation based on the identity of their N-terminal amino acids. Different N-end degrons are recognized by components of the degradative machinery allowing the proteins to be targeted by ATP-dependent proteases. In E. coli, the adaptor ClpS binds the N-degrons Leu, Ph, Tyr, and Trp and delivers proteins to the ClpAP complex. Proteins with N-terminal Lys and Arg acquire a Leu or Phe N-degron by the action of Aat, an aminoacyl tRNA protein transferase. We captured more than 100 proteins with N-degrons bound to ClpS. Virtually all of the proteins were N-terminally truncated. Many of the proteins had N-terminal Lys or Arg that had been modified by Leu/Phe aminoacyl transferase. By screening strains with mutations in over 50 genes annotated as proteases or peptidases, we identified peptidases responsible for cleavage of specific proteins. The sequences surrounding the N-degrons revealed motifs that appear to act as recognition sites for endoproteases. One hypothesis is that there are intrinsic sites cellular proteins that are targeted by proteases, generating cleaved products with N-end degrons. Such cleavage might be a cellular mechanism to alter the function of proteins and modify the biological activity of complexes in which the proteins participate. A second hypothesis is that endoproteases combined with ClpSAP are previously unappreciated participants in protein quality control and conduct constant surveillance of proteins to assess their functionality structural integrity. Studies with ClpP are focused on the mechanism of cell death that results from binding the acyldepsipeptide antibiotic ADEP and the structural changes needed for substrate entry into the degradation chamber. ADEP is an antibiotics made by Streptococcus hawaiiensis. When bound to ClpP ADEP activates indiscriminate degradation of partially unfolded proteins. ADEPs are being developed as novel antibiotics to target human pathogens. Current research is focused on the primary sequence and structural features of ClpP involved in binding ADEP and in the allosteric changes in ClpP that open the axial channel. The project has added importance because the site of ADEP binding is also the docking site for ClpX and ClpA. We screened a library of randomly mutagenized ClpP to identify mutants of ClpP that are insensitive to ADEP but retain activity of ClpP with its cognate ATPases. These mutants should be very rare and will identify sites in ClpP for ADEP or ClpX binding or involved in allosteric communication between functional sites. To ensure that the mutagenized ClpP retains activity, we designed a plasmid that conditionally expresses a toxic protein that must be degraded by ClpXP for cell survival. We isolated 8 different mutants of ClpP with the desired properties and are characterizing the mutants. All contain multiple mutations, and we are separating individual mutations to identify which of the alleles displays the observed phenotypes. These studies will provide insight onto the workings of ClpP and its interactions with different activating ligands. Studies of Clp function have been hindered by the lack of inhibitors that can be added to cell cultures to inhibit ClpP. Divalent Zn inhibits ClpP, and we have obtained a crystal structure of ClpP and identified the sites at which Zn binds. Zn is chelated by two critical residues that form the interface between subunits in the heptameric ring. Two catalytic residues, His122 and Asp171, also interact with the Zn. ClpP exists in two states, one with the handles interlaced to expand the degradation chamber and another with the handles in a collapsed state. The latter is either a latent state or a transient intermediate during the degradation cycle that allows product release. Zn promotes or stabilizes a collapsed state of ClpP. We will obtain a set of bis(benzimidazole) compounds from Prof. Holden Thorp at the University of North Carolina that can enhance Zn binding to specific serine proteases. Substituents attached to the core of the compound can greatly enhanced binding affinity and specificity, and we will screen a large number of such compounds to find an inhibitor with high affinity for ClpP. ClpP is essential for growth or for virulence for a number of human pathogens, including Mycobacterium tuberculosis. We are collaborating with the laboratory of Alfred Goldberg at Harvard Medical School, who has provided us with purified ClpP from M. 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 crystallized Mbt-ClpP and obtained a density map at about 3.6 Angstroms. We have confirmed that the structure contains mixed tetradecamers made up of ClpP1 and ClpP2 rings. We expect to have a structure of the native protein this year. The crystal structure should guide the design of small molecule inhibitors that will 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. More than 30 proteins are increased within 16 hours of depletion of hClpP with siRNA. Many of the proteins 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. Cisplatin accumulation increases when ClpP is knocked down, and there is a marked increase in cisplatin-mediated damage to mitochondrial DNA. We find 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, which are used by cisplatin to enter and exit cells. No changes were observed in the copper transporter Ctr1 when ClpP was over-expressed or knocked down, but there was a correlation between the levels of ClpP and the levels of the copper efflux pump, ATP7A. 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.
