Insulin-degrading enzyme hydrolyzes ATP

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.     

Insulin-degrading enzyme is exported via an unconventional protein secretion pathway

Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid β-protein (Aβ), and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD). Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s) remain poorly understood. To address this topic, we investigated the effects of known inhibitors or stimulators of protein secretion on the secretion of IDE from murine hepatocytes and HeLa cells. IDE secretion was found to be unaffected by the classical secretion inhibitors brefeldin A (BFA), monensin, or nocodazole, treatments that readily inhibited the secretion of α1-antitrypsin (AAT) overexpressed in the same cells. Using a novel cell-based Aβ-degradation assay, we show further that IDE secretion was similarly unaffected by multiple stimulators of protein secretion, including glyburide and 3'-O-(4-benzoyl)benzoyl-ATP (Bz-ATP). The calcium ionophore, A23187, increased extracellular IDE activity, but only under conditions that also elicited cytotoxicity. Our results provide the first biochemical evidence that IDE export is not dependent upon the classical secretion pathway, thereby identifying IDE as a novel member of the select class of unconventionally secreted proteins. Further elucidation of the mechanisms underlying IDE secretion, which would be facilitated by the assays described herein, promises to uncover processes that might be defective in disease or manipulated for therapeutic benefit.     

Natural regulatory mechanisms of insulin degradation by insulin degrading enzyme

Insulin-degrading enzyme (IDE) accounts for most of the insulin degrading activity in extracts of several tissues and plays an important role in the intracellular degradation of insulin. Using newly developed sandwich radioimmunoassay for rat IDE, this enzyme was detectable in all tissues we examined and liver had the highest level of IDE. The ratio of insulin degrading activity to IDE concentration was roughly the same in liver, brain and muscle, however, twice as high in kidney as compared with other tissues. On the contrary, its degrading activity in these tissue extracts, including kidney, was completely lost after immunoprecipitation of IDE. These results suggest that IDE degrades insulin in the initial step of cleavage and that there are some mechanisms to regulate insulin degrading activity by IDE in the tissues.     

降解烤烟秸秆和烟碱菌株的筛选及其产酶特性

摘要:【目的】为获得能够降解烤烟秸秆和烟碱的菌株,并探索其降解烤烟秸秆的利用途径。【方法】以烤烟秸秆为唯一碳氮源,从烟田土壤中进行了菌株的筛选。采用形态学观察、生理生化特性鉴定、16S rRNA基因序列鉴定等方法对该菌株进行了鉴定,并对其以烤烟秸秆为底物进行液态发酵的产酶活性和木质纤维素降解效果进行了测定。【结果】结果表明:该菌株为巨大芽孢杆菌(Bacillus megaterium)。在以烤烟秸秆为主要营养物质液态发酵条件下该菌株具有较强的木质素降解能力,最大漆酶活力达到418.52 U/L,而木质素过氧化物酶和锰过氧化物酶的最大酶活分别为19.71 U/L 和64.71 U/L。此外,发酵20 d后该菌能够完全降解发酵液中的烟碱。【结论】本研究筛选到了1株能够较好降解烤烟秸秆和完全降解烟碱的巨大芽孢杆菌(Bacillus megaterium),且该菌株具有利用烤烟秸秆生产漆酶的应用价值。     

Rat brain insulin degrading enzyme in insulin and thyroid hormones imbalances

Rat brain insulin degrading enzyme activity and its relationship with insulin receptor were investigated in experimental hyperglycemia, hyperinsulinemia, hypothyroidism and hyperthyroidism. Insulin degrading enzyme activity was assessed in synaptosomes and high speed cytosol using [125I]insulin. Levels of insulin degrading enzyme were changed in high speed cytosol in insulin and thyroid hormone imbalances. These results suggest that insulin degrading enzyme in brain is predominantly active in cytosol and is subject to regulation by insulin and thyroid hormones. Probably it plays some role in long term effects of insulin in brain.     

The C-terminal domain of human insulin degrading enzyme is required for dimerization and substrate recognition

Insulin degrading enzyme (IDE), a zinc metalloprotease, can specifically recognize and degrade insulin, as well as several amyloidogenic peptides such as amyloid beta (Abeta) and amylin. The disruption of IDE function in rodents leads to glucose intolerance and cerebral Abeta accumulation, hallmarks of type 2 diabetes and Alzheimer's disease, respectively. Using limited proteolysis, we found that human IDE (113kDa) can be subdivided into two roughly equal sized domains, IDE-N and IDE-C. Oligomerization plays a key role in the activity of IDE. Size-exclusion chromatography and sedimentation velocity experiments indicate that IDE-N is a monomer and IDE-C serves to oligomerize IDE-N. IDE-C alone does not have catalytic activity. It is IDE-N that contains the crucial catalytic residues, however IDE-N alone has only 2% of the catalytic activity of wild type IDE. By complexing IDE-C with IDE-N, the activity of IDE-N can be restored to approximately 30% that of wild type IDE. Fluorescence polarization assays using labeled insulin reveal that IDE-N has reduced affinity to insulin relative to wild type IDE. Together, our data reveal the modular nature of IDE. IDE-N is the catalytic domain and IDE-C facilitates substrate recognition as well as plays a key role in the oligomerization of IDE.     

How the binding and degrading capabilities of insulin degrading enzyme are affected by ubiquitin

Insulin degrading enzyme (IDE) is known to play a pivotal role on amyloidogenic peptide degradation but little is known about the changes in the proteolytic activity of the enzyme upon modification of external factors. Particularly, although it has been reported that altered ubiquitin concentration and/or hyperinsulinaemia increase the risk of developing Alzheimer's disease (AD), the molecular mechanism involved is unclear. In this work, we study the role that ubiquitin plays on IDE capability of binding and degrading insulin molecules and the obtained results indicate that ubiquitin has an allosteric role for IDE and high ubiquitin levels impair IDE activity.     

Purification and properties of an enzyme capable of degrading the sheath of Sphaerotilus natans

Microorganisms which can degrade and grow on the purified sheath of a sheathed bacterium Sphaerotilus natans were collected from soil and river water. Two bacterial strains were isolated from the soil and designated strains TB and TK. Both strains are rod shaped, negatively stained by gram staining, facultatively anaerobic, and formed ellipsoidal endospores. These characteristics suggested that the isolates belong to the genus Paenibacillus, according to Ash et al. (C. Ash, F. G. Priest, and M. D. Collins, Antonie Leeuwenhoek 64:253-260, 1993). Phylogenetic analysis based on the 16S rDNA supported this possibility. Purification of the sheath-degrading enzyme was carried out from the culture broth of strain TB. The molecular weight of the enzyme was calculated to be 78,000 and 50, 000 by sodium dodecyl sulfate-polyacrylamide electrophoresis and gel filtration chromatography, respectively. Enzyme activity was optimized at pH 6.5 to 7.0 and 30 to 40 degrees C. The reaction was accelerated by the addition of Mg(2+), Ca(2+), Fe(3+), and iodoacetamide, whereas it was inhibited by the addition of Cu(2+), Mn(2+), and dithiothreitol. The enzyme acted on the polysaccharide moiety of the sheath, producing an oligosaccharide the size of which was between the sizes of maltopentaose and maltohexaose. As the reaction proceeded, the absorbance at 235 nm of the reaction mixture increased, suggesting the generation of unsaturated sugars. Incorporation of unsaturated sugars was also suggested by the thiobarbituric acid reaction. It is possible that the enzyme is not a hydrolytic enzyme but a kind of polysaccharide eliminase which acts on the basic polysaccharide.     

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. Sequencing of the B chain fragments demonstrated that the Drosophila enzyme cleaves the B chain of insulin at four sites between residues B10 and B11, B14 and B15, B16 and B17, and B25 and B26. These cleavage sites correspond to four of the seven cleavage sites generated by the mammalian insulin protease. These results demonstrate that all the insulin cleavage sites generated by the Drosophila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.     

Identification of the metal associated with the insulin degrading enzyme.

Insulin degrading enzyme (IDE) is a thiol-dependent metalloendoprotease that is responsible for initiation of cellular insulin degradation. However, its exact mode of action and the factors controlling it are poorly understood. Since IDE is a metal requiring enzyme, we have examined which metal(s) is(are) endogenously associated with it. Using neutron activation analysis, we studied the metal content of a partially purified enzyme from three different tissues: rat skeletal muscle, rat liver, and human placenta. Our results indicate that zinc and manganese are associated with the enzyme with approximately 10 times more zinc as manganese being present. These results suggest that one or both of these two metals are endogenously associated with this enzyme and are a means of controlling the enzyme's activity.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response

Natriuretic peptides (NPs) are cyclic vasoactive peptide hormones with high therapeutic potential. Three distinct NPs (ANP, BNP, and CNP) can selectively activate natriuretic peptide receptors, NPR-A and NPR-B, raising the cyclic GMP (cGMP) levels. Insulin-degrading enzyme (IDE) was found to rapidly cleave ANP, but the functional consequences of such cleavages in the cellular environment and the molecular mechanism of recognition and cleavage remain unknown. Here, we show that reducing expression levels of IDE profoundly alters the response of NPR-A and NPR-B to the stimulation of ANP, BNP, and CNP in cultured cells. IDE rapidly cleaves ANP and CNP, thus inactivating their ability to raise intracellular cGMP. Conversely, reduced IDE expression enhances the stimulation of NPR-A and NPR-B by ANP and CNP, respectively. Instead of proteolytic inactivation, IDE cleavage can lead to hyperactivation of BNP toward NPR-A. Conversely, decreasing IDE expression reduces BNP-mediated signaling. Additionally, the cleavages of ANP and BNP by IDE render them active with NPR-B and a reduction of IDE expression diminishes the ability of ANP and BNP to stimulate NPR-B. Our kinetic and crystallographic analyses offer the molecular basis for the selective degradation of NPs and their variants by IDE. Furthermore, our studies reveal how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to biased stochastic, non-sequential cleavages and the ability of IDE to switch its substrate selectivity. Thus, the evolutionarily conserved IDE may play a key role in modulating and reshaping the strength and duration of NP-mediated signaling.     

Insulin-degrading enzyme: embarking on amyloid destruction.

Several human disorders are caused by or associated with the deposition of protein aggregates known as amyloid fibrils. Despite the lack of sequence homology among amyloidogenic proteins, all amyloid fibrils share a common morphology, are insoluble under physiological conditions and are resistant to proteolytic degradation. Because amyloidogenic proteins are being produced continuously, eukaryotic organisms must have developed a form of proteolytic machinery capable of controlling these aggregation-prone species before their fibrillization. This article suggests that an intracellular metalloprotease called insulin-degrading enzyme (IDE) is responsible for the elimination of proteins with amyloidogenic potential and proposes a mechanism for the selectivity of the enzyme. In this respect, IDE can also be referred to as ADE: amyloid-degrading enzyme.     

Purification and properties of an enzyme capable of degrading the polysaccharide of the cyanobacterium,Nostoc commune

A novel Nostoc commune-polysaccharide (NPS)-degrading enzyme with a molecular mass of 128.5 kDa was purified from Paenibacillus glycanilyticus DS-1. The optimum pH and temperature of the enzyme activity were 5.5 and 35 degrees C, respectively. The enzyme completely degraded NPS to oligosaccharides, ranging from tetra to hexasaccharides and could degrade the xylan weakly whereas xanthan, gellan, cellulose, curdlan and p-nitrophenyl-beta-D-xylopyranoside were not degraded. Homology analysis of the N-terminal amino acid sequence of the NPS-degrading enzyme against the PIR and SWISS-PROT databases indicated that the sequence was not homologous to any other polysaccharide-degrading enzyme.     

The biology of aerobic methylobacteria capable of degrading halomethanes

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyletrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH2O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and protection of the biosphere from these toxic pollutants.     

Characterization of a microbial consortium capable of degrading lignocellulose

A microbial consortium, designated WCS-6, was established by successive subcultivation in the presence of rice straw under static conditions. The degradation efficiencies of WSC-6 for 0.5 g filter paper, cotton and rice straw after 3 days of cultivation were 99.0±0.7%, 76.9±1.5% and 81.3±0.8%, respectively as determined by gravimetrical methods. Nine bacterial isolates were obtained from WCS-6 plated under aerobic conditions, and sequencing of their 16S rDNA indicated that these bacteria were related to Bacillus thermoamylovorans BTa, Paenibacillus barengoltzii SAFN-016, Proteobacterium S072, Pseudoxanthomonas taiwanensis CB-226, Rhizobiaceae str. M100, Bacillus sp. E53-10, Beta proteobacterium HMD444, Petrobacter succinimandens 4BON, and Tepidiphilus margaritifer N2-214. DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rDNA sequences amplified from total consortium DNA revealed the presence of sequences related to those of Ureibacillus thermosphaericus, uncultured bacterium clone GC3, uncultured Clostridium sp. clone A1-3, Clostridium thermobutyricum, and Clostridium thermosuccinogenes in addition to the sequences identified from the cultured bacteria. The microbial community identified herein is a potential candidate consortium for the degradation of waste lignocellulosic biomass.     

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.     

微生物降解石油烃的功能基因研究进展

微生物对石油烃的降解在自然衰减去除土壤和地下水石油烃污染的过程中发挥了重要作用。微生物通过其产生的一系列酶来利用和降解这类有机污染物,其中,编码关键降解酶的基因称为功能基因。功能基因可作为生物标志物用于分析环境中石油烃降解基因的多样性。因此,研究石油降解功能基因是分析土著微生物群落多样性、评价自然衰减潜力与构建基因工程菌的重要基础。本文主要介绍了烷烃和芳香烃在有氧和无氧条件下的微生物降解途径,重点总结了烷烃和芳香烃降解的主要功能基因及其作用,包括参与羟化作用的单加氧酶和双加氧酶基因、延胡索酸加成反应的琥珀酸合酶基因以及中心中间产物的降解酶基因等。