A single step purification for autolytic zinc proteinases

We describe a novel single-step method for the purification of stromelysin-1 catalytic domain (SCD) via immobilized metal affinity chromatography under denaturing conditions that inhibit proteolytic activity followed by on-column refolding and spontaneous autolysis of the fusion peptide to yield pure, active stromelysin-1 catalytic domain. The methodology provides a general approach for the rapid purification of large quantities of zinc proteinases.     

Independent refolding of domains in the pancreatic serine proteinases

Ser-neotrypsinogen and Val-neotrypsinogen are two-chain modifications of bovine trypsinogen produced on limited proteolysis with trypsin. Ser-neotrypsinogen has Lys131-Ser132 cleaved in the connecting peptide (the autolysis loop) linking the amino- and carboxyl-terminal domains. Val-neotrypsinogen has Arg105-Val106 cleaved which is located within the amino-terminal domain. The mixed disulfide derivative of Ser-neotrypsinogen was successfully refolded. A functional molecule was regenerated from the polypeptide fragments with the correct molecular weight of neotrypsinogen in an overall yield of 7%. Val-Neotrypsinogen could not be refolded. The first-order rate constants for the regeneration of Ser-neotrypsinogen were determined from the formation of active enzyme molecules as a function of time and from the regain of the correct molecular weight. Both kinetic values were the same indicating that refolding of the polypeptide chains first forms globular domain structures. The two domains then associate and the disulfide bonds between the domains and the correct geometry of the active site residues are formed last. The same kinetic results were also found in refolding Thr-neochymotrypsinogen (Duda, C. T., and Light, A. (1982) J. Biol. Chem. 257, 9866-9871) where peptide bond cleavage also occurred in the connecting peptide. These observations support the hypothesis that the pathway of folding of serine proteinases proceeds with the independent refolding of domains.     

New Aspartic Proteinase of Ulysses Retrotransposon from Drosophila virilis

This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.     

A hydrophobic cluster forms early in the folding of dihydrofolate reductase

The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site-directed mutagenesis and by physicochemical means. The absence of this fluorescence-detected phase in the refolding of the Trp-74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp-74. In addition, water-soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and native-like conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp-74 from solvent.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

Early intermediates in the PDI-assisted folding of ribonuclease A

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.     

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

Mutants of plasminogen activator inhibitor-1 designed to inhibit neutrophil elastase and cathepsin G are more effective in vivo than their endogenous inhibitors

Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.     

Early steps in the refolding reaction of reduced ribonuclease A.

The early part of the reaction of refolding of reduced ribonuclease A has been studied using the reappearance of enzymatic activity as an index of refolding. It is found that a low level of activity, about 0.04% of that of native enzyme, can be measured early after refolding has been initiated. This low level of activity is apparently not due to a contaminant or to incompletely reduced RNAase A molecules, but rather seems to be a property of the bulk of the reduced protein. Furthermore, this early activity is sensitive to the reaction with N-ethyl-maleimide, showing that it is due to completely or partially reduced molecules. The amount of protein responsible for this early activity represents a small fraction of the total reduced RNAase A, and possesses binding properties similar to those of the native enzyme towards a substrate, 2′, 3′ CMP and an inhibitor, 2′ CMP. These results are interpreted as evidence for the existence of an equilibrium between native and unfolded conformations in reduced RNAase A, and are discussed with respect to the protein folding mechanism.     

The role of Trp-82 in the folding of intestinal fatty acid binding protein

Multiple phases have been observed during the folding and unfolding of intestinal fatty acid binding protein (WT-IFABP) by stopped-flow fluorescence. Site-directed mutagenesis has been used to examine the role of each of the two tryptophans of this protein in these processes. The unfolding and refolding kinetics of the mutant protein containing only tryptophan 82 (W6Y-IFABP) showed that the tryptophan at this location was critical to the fluorescence signal changes observed throughout the unfolding reaction and early in the refolding reaction. However, the kinetic patterns of the mutant protein containing only tryptophan 6 (W82Y-IFABP) indicated that the tryptophan at this location participated in the fluorescence signal changes observed early in the unfolding reaction and late in the refolding reaction. Together, these data suggest that native-like structure was formed first in the vicinity of tryptophan 82, near the center of the hydrophobic core of this beta-sheet protein, prior to formation of native-like structure in the periphery of the protein.     

Structural insights into the activation of P. vivax plasmepsin

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.     

包含体蛋白质的复性研究进展

包含体的形成是异源蛋白质在大肠杆菌中高效表达的必然结果,也是目前产生重组蛋白质最有效的方法之一。不可溶、无生物活性的包含体必须经过变性、复性才能获得天然结构,完整特定的生物学功能。聚集是造成重组蛋白质复性产率低下的主要因素,因此理解蛋白质聚集机制,减少和防止聚集的发生是建立高效、高产率复性方法的关键。分子伴侣、低分子量添加物等在复性过程中的应用及新的复性方法的建立都大大提高了重组蛋白质复性产率。     

Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants

A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.     

Monoclonal antibody epitope mapping describes tailspike beta-helix folding and aggregation intermediates

There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.     

金鱼不同组织器官及胚胎发育不同时期蛋白水解酶的种类和活性变化

采用复性电泳方法研究了金鱼组织器官蛋白水解酶及个体发生过程中蛋白水解酶的种类和活性变化,主要结果表明：⑴金鱼各组织器官蛋白水解酶种类差异不大,大多数组织器官都具有１１３、６９、２０、１６ｋＤ四条带,但不同组织器官常具有其特异性蛋白水解酶;肠道蛋白水解酶种类最多、活性最强。⑵蛋白水解酶的活性受ｐＨ值影响和制约,大多数组织器官蛋白水解酶活性最适ｐＨ值为８．５。⑶在金鱼胚胎发育早期（从卵裂到心跳期）多数     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

Effect of low-temperature hardening on activities of proteolytic enzymes and their inhibitors in the leaves of wheat and cucumber seedlings

On seedlings of winter wheat (Triticum aestivum L.) and cucumber (Cucumis sativus L.), the dynamics of cysteine and serine trypsin-like proteinases and also trypsin inhibitors at cold hardening (5°C for wheat and 10°C for cucumber) was studied. Activation of proteinases and inhibitors coincided in time or preceded an increased tolerance in wheat and cucumber seedlings in the early period of their hardening. After attaining the highest wheat tolerance, activity amidases reduced, whereas the increased activity levels of cysteine proteinases and trypsin inhibitors was maintained during the entire period of hardening. In cucumber, in these period activities of amidases and trypsin inhibitors reduced, whereas the activity of cysteine proteinases was maintained at the level close to the initial one. It is suggested that cysteine proteinases, amidases, and trypsin inhibitors are involved in plant adaptation to cold.     

The role of proteolytic processes in the stimulation of lipolysis in the adipose tissue by somatotropin, adrenocorticotropin and beta-lipotropin

The influence of proteinase inhibitors on the lipotropic effect of somatotropic (STH), adrenocorticotropic (ACTH) and beta-lipotropic (LPH) hormones in adipose tissue was studied in vitro. The effect of STH was found to be completely dependent on the activity of tissue serine proteinases of trypsin and chymotrypsin types. The effect of LPH partly depended on serine proteinases of chymotrypsin type, whereas that of ACTH--on chymotrypsin and carboxylic proteinases. The effects of all the three hormones were also manifested during lysosomal proteolysis. The protease-dependent inhibition was specific for polypeptide hormones and was unobserved in the lipotropic effect of adrenaline. The inhibiting effect of serine proteinase inhibitors on hormones pretreated with blood plasma or proteinases was much weaker than on untreated hormones. In adipose tissue the early insulin-like effect of STH, unlike the late lipotropic effect, was independent of proteolysis. It was assumed that primary proteolysis plays a role in the activation of polypeptide hormones which is necessary for the manifestation of the lipotropic action.     

Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions

The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can enter two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.     

Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites

Acanthamoeba species can cause granulomatous encephalitis and keratitis in man. The mechanisms that underlie tissue damage and invasion by the amoebae are poorly understood, but involvement of as yet uncharacterized proteinases has been suggested. Here, we employed gelatin-containing gels and azocasein assays to examine proteinase activities in cell lysates and in medium conditioned by Acanthamoeba polyphaga trophozoites. Azocasein hydrolysis by cell lysates was optimally detected at pH 4.0-5.0 and was predominantly associated with the activity of cysteine proteinases. Compatible with enzyme activation during secretion, culture supernatants additionally contained a prominent azocasein hydrolyzing activity attributable to serine proteinases; these enzymes were better detected at pH 6.0 and above, and resolved at 47, 60, 75, 100, and >110 kDa in overlay gelatin gels. Although a similar banding profile was observed in gels of trophozoite lysates, intracellular serine proteinases were shown to be activated during electrophoresis and to split the substrate during migration in sodium dodecyl sulfate gels. Blockage of serine proteinases with phenylmethylsulfonylfluoride prior to electrophoresis permitted the detection of 43-, 59-, 70-, and 100-130-kDa acidic cysteine proteinases in cell lysates, and of 3 (43, 70, and 130 kDa) apparently equivalent enzymes in culture supernatants. Under the conditions employed, no band associated with a metalloproteinase activity could be depicted in substrate gels, although the discrete inhibition of supernatants' azocaseinolytic activity by 1,10-phenanthroline suggested secretion of some metalloproteinase.