The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity

The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.     

Specificity of the wound-induced leucine aminopeptidase (LAP-A) of tomato activity on dipeptide and tripeptide substrates.

Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A) that preferentially hydrolyzes amino acid-p-nitroanilide and -beta-naphthylamide substrates with N-terminal Leu, Met and Arg residues. To determine the substrate specificity of LAP-A on more natural substrates, the rates of hydrolysis of 60 dipeptide and seven tripeptide substrates were determined. For comparison, the specificities of the porcine and Escherichia coli LAPs were evaluated in parallel. Several marked differences in substrate specificities for the animal, plant and prokaryotic LAP enzymes were observed. Substrates with variable N-terminal (P1) residues (Xaa) were evaluated; these substrates had Leu or Gly in the penultimate (P1') position. The plant, animal, and prokaryotic LAPs hydrolyzed dipeptides with N-terminal nonpolar aliphatic (Leu, Val, Ile, and Ala), basic (Arg), and sulfur-containing (Met) residues rapidly, while P1 Asp or Gly were cleaved inefficiently from peptides. Significant differences in the cleavage of dipeptides with P1 aromatic residues (Phe, Tyr, and Trp) were noted. To systematically evaluate the impact of the P1' residue on cleavage of dipeptides, three series of dipeptides (Leu-Xaa, Gly-Xaa, and Arg-Xaa) were evaluated. The P1' residue strongly influenced hydrolysis of dipeptides and the magnitude of its effect was dependent on the P1 residue. P1' Pro, Asp, Lys and Gly slowed the hydrolysis rates of the tomato LAP-A, porcine LAP, and E. coli PepA markedly. Analysis six Arg-Gly-Xaa tripeptides showed that more diversity was tolerated in the P2' position. P2' Arg inhibited tripeptide cleavage by all three enzymes, while P2' Asp enhanced hydrolysis rates for the porcine and prokaryotic LAPs.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

20S proteasomes have the potential to keep substrates in store for continual degradation

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.     

Force spectroscopy of substrate molecules en route to the proteasome's active sites

We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (β-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome.     

DNA-catalyzed covalent modification of amino acid side chains in tethered and free peptide substrates

This study focuses on the development of DNA catalysts (deoxyribozymes) that modify side chains of peptide substrates, with the long-term goal of achieving DNA-catalyzed covalent protein modification. We recently described several deoxyribozymes that modify tyrosine (Tyr) or serine (Ser) side chains by catalyzing their reaction with 5'-triphosphorylated RNA, forming nucleopeptide linkages. In each previous case, the side chain was presented in a highly preorganized three-dimensional architecture such that the resulting deoxyribozymes inherently cannot function with free peptides or proteins, which do not maintain the preorganization. Here we describe in vitro selection of deoxyribozymes that catalyze Tyr side chain modification of tethered and free peptide substrates, where the approach can potentially be generalized for catalysis involving large proteins. Several new deoxyribozymes for Tyr modification (and several for Ser modification as well) were identified; progressively better catalytic activity was observed as the selection design was strategically changed. The best new deoxyribozyme, 15MZ36, catalyzes covalent Tyr modification of a free tripeptide substrate with a k(obs) of 0.50 h(-1) (t(1/2) of 83 min) and up to 65% yield. These findings represent an important advance by demonstrating, for the first time, DNA catalysis involving free peptide substrates. The new results suggest the feasibility of DNA-catalyzed covalent modification of side chains of large protein substrates and provide key insights into how to achieve this goal.     

Behavioural response of Kootenai white sturgeon (Acipenser transmontanus,Richardson, 1836) early life stages to gravel,pebble, and rubble substrates: guidelines for rearing substrate size

Guidelines for creating rearing substrate for sturgeon early life stages are needed for restoration programmes creating habitats for spawning and rearing of early life stages. To determine the effects of rock size on motile early life stages, experiments were conducted in artificial streams to observe the behaviour of free embryos and larvae of Kootenai River white sturgeon (Acipenser transmontanus) relative to rock size. Most (≥90%) of the free embryos in replicate test streams with 100% gravel, 100% pebble, or 100% rubble hid under rocks, with few moving downstream. There was no difference in downstream movement of free embryos among rock treatments, therefore all rock types provided cover habitat. Similarly, in rock mixture tests, with a variable percentage of pebble, small rubble, or large rubble in different tanks, even fewer free embryos moved downstream. With increasing age, larvae increasingly used the open bottom and velocity refuges downstream of or alongside rocks of any size while drift feeding. Downstream movement of larvae in both rock regime tests was affected by rock size, with significantly reduced movement relative to increasing abundance of large rock (rubble). However, in all rock mixtures, free embryos (and later, larvae when they stopped dispersing) preferred the smallest rock size available (pebble; P = 0.0001). This suggests a strong innate preference of both life stages for small substrate that is likely related to increased survival. A rock mixture of 10% gravel (16–32 mm diameter) and 30–40% pebble (diameter, 30–60 mm) should provide adequate rearing substrate for free embryos and early‐larvae. The remaining 50–60% should be mixed rubble and boulders for spawning and egg rearing.     

Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.     

Role of substrate in reversible activation of proteasomes (multi-protease complexes) by sodium dodecyl sulfate

Previously, we reported that proteasomes (large multi-protease complexes) are present in a latent state in a variety of eukaryotic cells, and can be activated by treatment with various compounds such as sodium dodecyl sulfate (SDS) or poly-lysine (Tanaka et al. (1988) J. Biol. Chem. 263, 16209-16217). In the present study, the mechanism of activation of latent proteasomes by SDS was examined. Latent proteasomes were greatly activated by addition of low concentrations of 0.04 to 0.08% SDS in the presence of substrate. This activation appeared to be reversible, because SDS-activated proteasomes returned to a latent state when the concentration of SDS was reduced by dilution. In contrast, in the absence of substrate, latent proteasomes lost their activity almost completely in an irreversible fashion within a few minutes during treatment with SDS at either 0 or 37 degrees C. Interestingly, SDS-treated proteasomes were markedly protected against this rapid inactivation by either a peptide or protein substrate. Moreover, removal of the substrate after activation of proteasomes caused their rapid irreversible inactivation. These results indicate that the substrate is necessary for reversible activation of latent proteasomes by SDS. This effect of substrate is presumably important in regulation of intracellular protein breakdown by activated proteasomes in eukaryotic cells.     

26 S proteasomes function as stable entities.

Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.     

The Proteasome-associated Protein Ecm29 Inhibits Proteasomal ATPase Activity and in Vivo Protein Degradation by the Proteasome

Several proteasome-associated proteins regulate degradation by the 26 S proteasome using the ubiquitin chains that mark most substrates for degradation. The proteasome-associated protein Ecm29, however, has no ubiquitin-binding or modifying activity, and its direct effect on substrate degradation is unclear. Here, we show that Ecm29 acts as a proteasome inhibitor. Besides inhibiting the proteolytic cleavage of peptide substrates in vitro, it inhibits the degradation of ubiquitin-dependent and -independent substrates in vivo. Binding of Ecm29 to the proteasome induces a closed conformation of the substrate entry channel of the core particle. Furthermore, Ecm29 inhibits proteasomal ATPase activity, suggesting that the mechanism of inhibition and gate regulation by Ecm29 is through regulation of the proteasomal ATPases. Consistent with this, we identified through chemical cross-linking that Ecm29 binds to, or in close proximity to, the proteasomal ATPase subunit Rpt5. Additionally, we show that Ecm29 preferentially associates with both mutant and nucleotide depleted proteasomes. We propose that the inhibitory ability of Ecm29 is important for its function as a proteasome quality control factor by ensuring that aberrant proteasomes recognized by Ecm29 are inactive.     

β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism

1. beta-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000-150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris-HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The K(m) was 3.9x10(-4)m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the beta-ketothiolase condensation reaction and its possible involvement in the regulation of poly-beta-hydroxybutyrate synthesis and degradation are discussed.     

Dependence of proteasome processing rate on substrate unfolding

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ～5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.     

New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay

Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.     

Dissecting various ATP-dependent steps involved in proteasomal degradation

Proteolysis by archaeal 20S proteasomes and the PAN (proteasome-activating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S AAA ATPases, requires ATP hydrolysis through multiple steps. ATP hydrolysis, activated by binding of substrates to PAN, is utilized for substrate unfolding, gate opening of 20S proteasomes, and substrate translocation.     

Gamma-glutamyl hydrolase conjugase). Purification and properties of the bovine hepatic enzyme.

Bovine hepatic gamma-glutamyl hydrolase (conjugase) has been purified to homogeneity. A feature of the purification procedure was the use of high affinity macromolecular polyanion enzyme inhibitors which formed tight complexes with the enzyme altering its solubility, gel filtration, and ion exchange properties. The enzyme, which cleaves the gamma-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with its lysosomal localization. Zinc is essential for enzyme stability. The presence of highly reactive sulfhydryl groups was evident from the extreme sensitivity to oxidizing agents and organomercurials. Very little thermal denaturation occurs below 65 degrees, but the enzyme is extremely sensitive to 0uffer anions, in keeping with the polyanionic nature of the substrate. In order to study the mechanism of action of the enzyme, a wide range of pteroylpolyglutamates, N-t-Boc polyglutamates and free polyglutamates were synthesized containing L-[U-14C]glutamic acid residues in different positions. Two pteroyltriglutamate derivatives were also synthesized in which an alpha bond replaced one of the two available gamma bonds. Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at gamma-glutamyl bonds and the presence of a COOH-terminal gamma bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should therefore be more appropriately classified as an acid hydrolase; (c) longer chain gamma-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of gamma bonds was independent of the NH2-terminal pteroyl moiety. Studies with polyanions such as the glycosaminoglycans and dextran sulfate supported the concept that the polyanion structure of the substrate was a major factor in substrate-active site interaction.     

Two continuous spectrophotometric assays for methionine aminopeptidase

Two spectrophotometric assays have been developed for methionine aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 (k(cat)/K(M) = 2.8 x 10(5) M(-1) s(-1) for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a prolyl aminopeptidase reaction using Met-Pro-p-nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces prolyl-p-nitroanilide, which is rapidly hydrolyzed by the prolyl aminopeptidase from Bacillus coagulans to release a chromogenic product, p-nitroaniline. This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors.     

Fluorescence polarization for monitoring ribozyme reactions in real time

Fluorescence polarization has been used recently to monitor diverse macromolecular interactions. In this report, the application of fluorescence polarization has been extended to monitor ribozyme reactions in real time. With fluorescently labeled substrate RNAs, group I ribozyme ligation and hammerhead ribozyme cleavage reactions were studied by fluorescence polarization in substrate excess (multiple turnover) conditions. These results also show that fluorescently labeled RNAs remain active substrates for ribozymes. Furthermore, a direct comparison of fluorescence polarization with fluorescence resonance energy transfer showed that both techniques were comparable for monitoring ribozyme reactions.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.