ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific substrate of yeast metacaspase

Yeast metacaspase (Yca1p) is required for the execution of apoptosis upon a wide range of stimuli. However, the specific degradome of this yeast protease has not been unraveled so far. By combining different methodologies described as requisites for a protein to be considered a protease substrate, such as digestome analysis, cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, we show that upon H(2)O(2)-induced apoptosis, the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a specific target of metacaspase. Nitric oxide (NO) signaling, which mediates H(2)O(2)-induced apoptosis, is required for metacaspase specific GAPDH cleavage. In conclusion, in this work we identified GAPDH as the first direct yeast metacaspase substrate described so far. Although mammalian caspases and yeast metacaspase apparently have distinct target cleavage sites, GAPDH arises as a common substrate for these proteases.     

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.     

Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.     

Degradation of the precursor of mitochondrial aspartate aminotransferase in chicken embryo fibroblasts

The precursor of mitochondrial aspartate aminotransferase accumulates in the cytosol of cultured chicken embryo fibroblasts if its import into mitochondria is inhibited by an uncoupling agent. However, its accumulation is limited by degradation with a half-life of only approximately 5 min (Jaussi, R., Sonderegger, P., Flückiger, J., and Christen, P. (1982) J. Biol. Chem. 257, 13334-13340). The aim of the present study was the characterization of the proteolytic system(s) responsible for this very rapid intracellular degradation. On depleting chicken embryo fibroblasts of ATP, the rate of degradation of the precursor was lowered by approximately 70%. Chicken embryo fibroblasts depleted of divalent metal ions showed a degradative activity of 10% of the initial value. Reconstitution of these cells with Mg2+ and Ca2+ increased the degradative activity from 10 to 107 and 24%, respectively. Thiol reagents almost completely prevented the degradation, whereas specific peptide inhibitors of cysteine proteases or inhibitors of intralysosomal proteolysis decreased the rate of degradation by only approximately 30%. Inhibitors of serine proteases had little effect. No rapid degradation of the precursor was observed in crude extracts of chicken embryo fibroblasts. The data indicate that the bulk of the precursor accumulated under conditions of import block is degraded by one or several cytosolic proteases dependent on ATP, Mg2+, and thiol groups of unknown localization, conceivably by proteolytic enzymes identical with or similar to one of the high molecular weight cytosolic proteases (Waxman, L., Fagan, J.M., Tanaka, K., and Goldberg, A. L. (1985) J. Biol. Chem. 260, 11994-12000). The rest of the precursor appears to be degraded by lysosomes.     

Opposing effects of DNA on proteolysis of a replication initiator

DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.     

Modifications of glyceraldehyde-3-phosphate dehydrogenase induced by increasing concentrations of peroxynitrite: early recognition by 20S proteasome

Peroxynitrite, a potent oxidizing and nitrating species, induces covalent modifications of biomolecules in a number of pathological conditions. In previous studies with S. cerevisiae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as being especially susceptible to nitration by peroxynitrite. The activity of this enzyme was strongly inhibited by low doses of peroxynitrite in yeast and in cultured rat astrocytes. Here, the sequence of modifications of isolated mammalian GAPDH induced by increasing concentrations of peroxynitrite is demonstrated to be as follows: (i) oxidation, leading to inactivation and to enhanced susceptibility of GAPDH for proteasomal degradation, (ii) oligomer formation, and (iii) nitration. In our study the susceptibility for degradation by isolated 20S proteasome was by far the most sensitive parameter for peroxynitrite-induced damage to GAPDH, implying that this might also occur under pathological conditions where peroxynitrite is generated at low concentrations in vivo.     

A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in a Bacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria-Bertani (LB) medium, B. cereus contains a major NAD+-dependent GAPDH activity and only traces of NADP+-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP+-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show that B. cereus possesses two GAPDH activities, namely NAD+- and NADP+-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP+-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability.     

Partial Characterization of the Protease(s) Involved in the Degradation of Phytochrome Polypeptide in Etiolated Epicotyl Tissue of Pisum sativum

Effects of chelators and inhibitors of proteases and ATP-generatingsystems on the red-light-induced degradation of phytochromewere examined in apical segments of epicotyl from 7-day-old,etiolated pea seedlings. The results suggest that a serine protease(s)is involved in degradation of phytochrome, and that the protease(s)and/or the degradation process requires Fe²⁺ and/or Zn²⁺ andATP. (Received February 5, 1990; Accepted September 3, 1990)     

Direct Binding of Glyceraldehyde 3-Phosphate Dehydrogenase to Telomeric DNA Protects Telomeres against Chemotherapy-Induced Rapid Degradation

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel-shift assays, we show that recombinant GAPDH binds directly with high affinity (K_d = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats, and that nucleotides T1, G5, and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA:GAPDH), and GAPDH appears to form a high-molecular-weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active-site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth-inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition; this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.     

Proteolysis mediated by the membrane‐integrated ATP‐dependent protease FtsH has a unique nonlinear dependence on ATP hydrolysis rates

ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane‐integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40–60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water‐soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.     

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.     

桉蝙蛾幼虫实时荧光定量PCR内参基因的筛选

本研究通过筛选桉蝙蛾Endoclita signifer Walker幼虫不同龄期与不同体节中稳定表达的内参基因,为桉蝙蛾基因表达研究提供参考。通过实时荧光定量PCR技术测定5个候选内参基因（ACTIN、GAPDH、TUB、RIB、EF）在桉蝙蛾幼虫不同龄期（3龄、5龄、9龄、12龄）与不同体节（头部、胸部、腹部）及全样品（由所有样品组成）处理中的表达量,后利用GeNorm、NormFinder、BestKeeper对5个候选基因的稳定性进行评估,最后由RefFinder综合分析结果,选出最佳的内参基因。基于4种分析方法的评估可知,桉蝙蛾5龄和9龄幼虫的不同体节、不同龄期幼虫的头部和胸部中内参基因的最佳数目为2,而3龄和12龄幼虫的不同体节、不同龄期幼虫的腹部及全样本中内参基因的最佳数目为3。3龄、5龄、9龄和12龄幼虫的不同体节中可分别选择ACTIN+RIB+GAPDH、EF+RIB、GAPDH+EF和ACTIN+RIB+GAPDH作为最稳定的内参基因组合;EF+RIB、RIB+GAPDH和EF+GAPDH+ACTIN可分别作为不同龄期幼虫头部、胸部和腹部的最佳内参基因组合;综合考虑桉蝙蛾幼虫不同龄期与不同体节的影响时,可选择RIB、ACTIN和GAPDH作为内参基因。     

Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.     

Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation

Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.     

The interactions between soybean trypsin inhibitor and delta-endotoxin of Bacillus thuringiensis in Helicoverpa armigera larva

No significant difference in larval mortality was observed when a sublethal dose of Bacillus thuringiensis (Bt) var. kurstaki HD-1 crystal was supplemented with soybean trypsin inhibitor (STI) in the artificial diet fed to Helicoverpa armigera in the laboratory, but supplementing a nonlethal dose of crystal with STI in the diet led to a pronounced reduction of larval growth. This concentration of crystal and two lower concentrations of STI alone had no significant effects on larval growth. The results of substrate-gel electrophoresis demonstrated that the proteases in the H. armigera midgut fluid responsible for the degradation of protoxin consisted of at least four proteases with molecular weights of 71, 49, 36, and 30 kDa. All four proteases could utilize casein also as the substrate. When larvae were fed with STI or Bt + STI, the proteolytic activities of the 49-kDa enzyme disappeared, and the activities of the other three enzymes were reduced. Enzyme assays also indicated that feeding larvae with diets containing Bt, STI, or Bt + STI significantly decreased the specific activities of larval general proteases and the trypsin-like enzyme. The protein concentration of midgut fluid was elevated, especially in the larvae fed on the diets containing STI and Bt + STI. Both in vitro and in vivo studies showed that the degradation of protoxin and toxin could be inhibited by soybean trypsin inhibitors, but when the incubation time was prolonged, the protoxin could be degraded completely, while the degradation of toxin was inhibited further. This suggested that the retention time of toxins in the larval midgut was extended and synergism between insecticidal crystal protein and soybean trypsin inhibitor occurred, which showed as the inhibition of H. armigera larval growth.     

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.     

Localization of general and regulatory proteolysis in Bacillus subtilis cells

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.     

Interaction of Zn2+ and Cu2+ ions with glyceraldehyde-3-phosphate dehydrogenase from bovine heart and rabbit muscle.

1. Binding of Zn2+ and Cu2+ ions to GAPDHs from bovine heart and rabbit muscle resulted in a partial loss of enzymatic activity of both enzymes, in a time and metal ion concentration dependent manner. Cu2+ ions caused a much larger decrease of the activity than Zn2+ ions. 2. Addition of NAD+ or EDTA to either enzyme resulted in a protective effect on GAPDH activity. A similar protective effect was observed following addition of 2-mercaptoethanol to the enzyme solution. 3. The association constant for GAPDH-Zn2+ complex, calculated from equilibrium dialysis data, was 0.9 x 10(4) M-1 for the bovine heart GAPDH and 1.3 x 10(4) M-1 for the rabbit muscle enzyme. The association constant for GAPDH-Cu2+ complex was the same for both enzymes, 11.3 x 10(4) M-1. 4. Equilibrium dialysis data also revealed that in either enzyme the specific sites, binding the metal ions, are identical or very similar, and independent from each other. They are situated in the most conserved part of the enzyme molecule. 5. Some zinc was found in GAPDH preparations from bovine heart. It is discussed if Zn2+ ions could have a kind of modulation effect on GAPDH activity.     

Regulation of skeletal muscle myofibrillar protein degradation: relationships to fatigue and exercise

1. Exercise results in large alterations in cellular metabolic homeostasis and protein turnovers. Exhaustive exercise (as well as starvation, dystrophy, motor nerve disease) results in myofibrillar degradation and has been associated with the decreased force generating capabilities of muscle at fatigue. 2. Complete protein degradation is accomplished by the combined actions of non-lysosomal and lysosomal proteases and the initial breakdown of myofibrillar protein appears to be non-lysosomal mediated. 3. Current evidence suggests that covalent modification (mixed-function oxidation, formation of mixed disulfides, oxidation of methionine residues and phosphorylation) of proteins may mark them for degradation by rendering them more susceptible to proteolytic attack. 4. The rate of covalent modification can be controlled by the level of stabilizing and destabilizing ligands and by factors affecting the activity of the marking reaction. 5. The activities of individual proteases may be controlled by activators and inhibitors. 6. It is suggested that the large alterations in metabolism (hormonal profiles, energy status, redox status and Ca2+ levels) which accompany exercise serve to activate specific proteases and/or induce covalent modifications which mark specific myofibrillar proteins for subsequent proteolytic attack.