A Proteolytic Site-Directed Affinity Label to Inhibit the Human ATP-Dependent Protease Caseinolytic Complex XP.

Human caseinolytic protease component X and P (hClpXP) is a heterooligomeric ATP-dependent protease. The hClpX subunit catalyzes ATP hydrolysis whereas the hClpP subunit catalyzes peptide bond cleavage. In this study, we generated a peptidyl chloromethyl ketone (dansyl-FAPAL-CMK) that inhibited the hClpP subunit through alkylation of the catalytic His122, which was detected by LC-MS. This inhibitor is composed of a peptide sequence derived from a hydrolyzed peptide product of a substrate cleaved by hClpXP. Binding of FAPAL positions the electrophilic chloromethyl ketone moiety near His122 where alkylation occurs. Dansyl FAPAL-CMK exhibits selectivity for hClpXP over other ATP-dependent proteases such as hLon and the 26S proteasome and abolishes hClpXP activity in HeLa cell lysate. Using the fluorogenic peptide substrate FR-Cleptide as reporter, we detected biphasic inhibition time courses; this supports a slow-binding, time-dependent, covalent inhibition mechanism that is often found in active-site directed affinity labels. Because this inhibitor reacts only with hClpXP but not hLon or the proteasome, it has the potential to serve as a chemical tool to help validate endogenous protein substrates of hClpXP in cell lysate, thereby benefiting investigation of the physiological functions of hClpXP in different cell types or tissue samples.

Analysis of oxygen-18 labeled phosphate to study positional isotope experiments using LC-QTOF-MS.

A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography-QTOF-mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18O/16O, 31P and 15N isotope enrichment. Since these techniques either require a time-consuming derivatization step or have a limited sensitivity, an LC and accurate mass-based method for monitoring 18O/16O exchange was developed and compared with a standard GC-MS method. Our results showed that the LC-QTOF-MS method developed was not only as accurate as the standard GC-MS method, but also a sensitive and robust analytical platform for the simultaneous determination of isotope enrichment and the analysis of positional isotopes without chemical derivation. The LC-QTOF-MS method developed was successfully applied to the measurement of 18O/16O in the reversibility study of ATP hydrolysis by Lon proteases.

Utilization of Mechanistic Enzymology to Evaluate the Significance of ADP Binding to Human Lon Protease.

Lon, also known as Protease La, is one of the simplest ATP-dependent proteases. It is a homooligomeric enzyme comprised of an ATPase domain and a proteolytic domain in each enzyme subunit. Despite sharing about 40% sequence identity, human and Escherichia coli Lon proteases utilize a highly conserved ATPase domain found in the AAA+ family to catalyze ATP hydrolysis, which is needed to activate protein degradation. In this study, we utilized mechanistic enzymology techniques to show that despite comparable kcat and Km parameters found in the ATPase activity, human and E. coli Lon exhibit significantly different susceptibility to ADP inhibition. Due to the low affinity of human Lon for ADP, the conformational changes in human Lon generated from the ATPase cycle are also different. The relatively low affinity of human Lon for ADP cannot be accounted for by reversibility in ATP hydrolysis, as a positional isotope exchange experiment demonstrated both E. coli Lon and human Lon catalyzed ATP hydrolysis irreversibly. A limited tryptic digestion study however indicated that human and E. coli Lon bind to ADP differently. Taken together, the findings reported in this research article suggest that human Lon is not regulated by a substrate-promoted ADP/ATP exchange mechanism as found in the bacterial enzyme homolog. The drastic difference in structural changes associated with ADP interaction with the two protease homologs offer potential for selective inhibitor design and development through targeting the ATPase sites. In addition to revealing unique mechanistic differences that distinguish human vs. bacterial Lon, this article underscores the benefit of mechanistic enzymology in deciphering the physiological mechanism of action of Lon proteases and perhaps other closely related ATP-dependent proteases in the future.

CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+ Lon protease.

CODAS syndrome is a multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. Using whole-exome and Sanger sequencing, we identified four LONP1 mutations inherited as homozygous or compound-heterozygous combinations among ten individuals with CODAS syndrome. The individuals come from three different ancestral backgrounds (Amish-Swiss from United States, n = 8; Mennonite-German from Canada, n = 1; mixed European from Canada, n = 1). LONP1 encodes Lon protease, a homohexameric enzyme that mediates protein quality control, respiratory-complex assembly, gene expression, and stress responses in mitochondria. All four pathogenic amino acid substitutions cluster within the AAA(+) domain at residues near the ATP-binding pocket. In biochemical assays, pathogenic Lon proteins show substrate-specific defects in ATP-dependent proteolysis. When expressed recombinantly in cells, all altered Lon proteins localize to mitochondria. The Old Order Amish Lon variant (LONP1 c.2161C>G[p.Arg721Gly]) homo-oligomerizes poorly in vitro. Lymphoblastoid cell lines generated from affected children have (1) swollen mitochondria with electron-dense inclusions and abnormal inner-membrane morphology; (2) aggregated MT-CO2, the mtDNA-encoded subunit II of cytochrome c oxidase; and (3) reduced spare respiratory capacity, leading to impaired mitochondrial proteostasis and function. CODAS syndrome is a distinct, autosomal-recessive, developmental disorder associated with dysfunction of the mitochondrial Lon protease.

Characterization of E. coli manganese superoxide dismutase binding to RNA and DNA.

Bacterial manganese superoxide dismutase (MnSOD) has been shown to localize to the chromosomal portion of the cell and impart protection from ionizing radiation to DNA. The binding affinity of bacterial MnSOD to non-sequence specific double stranded oligomeric DNA has been quantitated previously by nitrocellulose filter binding and gel shift assays. In the current study we have examined the equilibrium binding of Escherichia coli MnSOD to poly(U), poly(A), poly(C), poly(dU) and double-stranded (ds) DNA. Equilibrium association constant, Kobs, was measured by monitoring intrinsic tryptophan fluorescence quenching. Based on the extent of quenching, Kobs was determined as a function of monovalent salt (MX) concentration and type, as well as temperature, from which ΔG°obs and ΔH°obs were determined. It was found that the polynucleotides bind to MnSOD in the following affinity hierarchy, poly(dU)>poly(U)>dsDNA>poly(A)>poly(C). The differences in the hierarchy were not large in magnitude as the poly(dU) bound with less than a 100-fold higher affinity than poly(C) at any given [MX]. For each polynucleotide, Kobs decreases only slightly with increasing [K(+)], surprising for a relatively non-specific nucleic acid protein. Thus, our finding that MnSOD can bind to RNA leads to the possibility that MnSOD may confer protection to RNA, as well. This is, as of yet, untested. Typically one would expect strong electrostatic interactions to dominate a non-specific binding event like that, but our results show an unexpectedly strong non-electrostatic contribution to the binding.

Processive degradation of unstructured protein by Escherichia coli Lon occurs via the slow, sequential delivery of multiple scissile sites followed by rapid and synchronized peptide bond cleavage events.

Processive protein degradation is a common feature found in ATP-dependent proteases. This study utilized a physiological substrate of Escherichia coli Lon protease known as the lambda N protein (λN) to initiate the first kinetic analysis of the proteolytic mechanism of this enzyme. To this end, experiments were designed to determine the timing of three selected scissile sites in λN approaching the proteolytic site of ELon and their subsequent cleavages to gain insight into the mechanism by which ATP-dependent proteases attain processivity in protein degradation. The kinetic profile of peptide bond cleavage at different regions of λN was first detected by the iTRAQ/mass spectrometry technique. Fluorogenic λN constructs were then generated as reporter substrates for transient kinetic characterization of the ATP- versus AMPPNP-dependent peptide bond cleavage and the delivery of the scissile sites near the amino- versus carboxyl-terminal of the λN protein to the proteolytic site of ELon. Collectively, our results support a mechanism by which the cleavage of multiple peptide bonds awaits the "almost complete" delivery of all the scissile sites in λN to the proteolytic site in an ATP-dependent manner. Comparing the time courses of delivery to the active site of the selected scissile sites further implicates the existence of a preferred directionality in the final stage of substrate delivery, which begins at the carboxyl-terminal. The subsequent cleavage of the scissile sites in λN, however, appears to lack a specific directionality and occurs at a much faster rate than the substrate delivery step.

Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity.

Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (K(λN)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(λN) values reveals that the region encompassing 232-252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation.