Wheat cultivars differing in heat tolerance show a differential response to monocarpic senescence under high-temperature stress and the involvement of serine proteases

High temperature is a common constraint during anthesis and grain-filling stages of wheat leading to huge losses in yield. In order to understand the mechanism of heat tolerance during monocarpic senescence, the present study was carried out under field conditions by allowing two well characterized Triticum aestivum L. cultivars differing in heat tolerance, Hindi62 (heat-tolerant) and PBW343 (heat-susceptible), to suffer maximum heat stress under late sown conditions. Senescence was characterized by measuring photosynthesis related processes and endoproteolytic activity during non-stress environment (NSE) as well as heat-stress environment (HSE). There was a faster rate of senescence under HSE in both the genotypes. Hindi62, having pale yellow flag leaf with larger area, maintained cooler canopy under high temperatures than PBW343. The tolerance for high temperature in Hindi62 was clearly evident in terms of slower green-leaf area degradation, higher stomatal conductance, higher stability in maximum PSII efficiency, Rubisco activity and Rubisco content than PBW343. Both the genotypes exhibited lower endopeptidase activity under HSE as compared to NSE and this difference was more apparent in Hindi62. Serine proteases are the predominant proteases responsible for protein degradation under NSE as well as HSE. Flag leaf of both the genotypes exhibited high-molecular-mass endoproteases (78 kDa and 67 kDa) isoforms up to full grain maturity which were inhibited by specific serine protease inhibitor in both the environments. In conclusion, the heat-tolerant Hindi62 exhibited a slower rate of senescence than the heat-susceptible PBW343 during HSE, which may contribute towards heat stability.     

Proteolytic activity and protease classes in the zooplankton species Calanus finmarchicus

The temperature optimum for the general proteolytic activity in the crude extract of Calanus finmarchicus was 50 degrees C and the pH optimum was found to be 7. The use of specific protease inhibitors resulted in the identification of at least three protease classes in the crude extract of C. finmarchicus. Those classes were serine, metallo and aspartic proteases. The serine and metallo proteases were found to be dominant under neutral to alkaline conditions and the aspartic proteases were dominant under acidic conditions. The cysteine proteases, if at all present, did not seem to be active or present in any substantial amount. The data presented points towards controlling and preserving the protein content in fresh C. finmarchicus.     

Expression and Localization of Phosphoenolpyruvate Carboxylase in Developing and Germinating Wheat Grains

Phosphoenolpyruvate carboxylase (PEPC) activity and corresponding mRNA levels were investigated in developing and germinating wheat (Triticum aestivum) grains. During grain development PEPC activity increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit remained almost constant throughout the process of grain development and in the scutellum and aleurone layer of germinating grains. The less-abundant 108-kD polypeptide progressively disappeared during the second half of grain development and was newly synthesized in the scutellum and aleurone layer of germinating grains. PEPC mRNA was detected throughout the process of grain development; however, in germinating grains PEPC mRNA accumulated transiently in the scutellum and aleurone layer, showing a sharp maximum 24 h after imbibition. Immunolocalization studies revealed the presence of the enzyme in tissues with a high metabolic activity, as well as in the vascular tissue of the crease area of developing grains. A clear increase in PEPC was observed in the scutellar epithelium of grains 24 h after imbibition. The data suggest that the transiently formed PEPC mRNA in the scutellar epithelium encodes the 108-kD PEPC subunit.     

A snapshot of the Ixodes scapularis degradome

Parasitic encoded proteases are essential to regulating interactions between parasites and their hosts and thus they represent attractive anti-parasitic druggable and/or vaccine target. We have utilized annotations of Ixodes scapularis proteases in gene bank and version 9.3 MEROPS database to compile an index of at least 233 putatively active and 150 putatively inactive protease enzymes that are encoded by the I. scapularis genome. The 233 putatively active protease homologs hereafter referred to as the degradome (the full repertoire of proteases encoded by the I. scapularis genome) represent ~1.14% of the 20485 putative I. scapularis protein content. Consistent with observations in other animals, the content of the I. scapularis degradome is ~6.0% (14/233) aspartic, ~19% (44/233) cysteine, ~40% (93/233) metallo, ~28.3% (66/233) serine and ~6.4% (15/233) threonine proteases. When scanned against other tick sequences, ~11% (25/233) of I. scapularis putatively active proteases are conserved in other tick species with ≥ 60% amino acid identity levels. The I. scapularis genome does not apparently encode for putatively inactive aspartic proteases. Of the 150 putative inactive protease homologs none are from the aspartic protease class, ~8% (12/150) are cysteine, ~58.7% (88/150) metallo, 30% (45/150) serine and ~3.3% (5/150) are threonine proteases. The I. scapularis tick genome appears to have evolutionarily lost proteolytic activity of at least 6 protease families, C56 and C64 (cysteine), M20 and M23 (metallo), S24 and S28 (serine) as revealed by a lack of the putatively active proteases in these families. The overall protease content is comparable to other organisms. However, the paucity of the S1 chymotrypsin/trypsin-like serine protease family in the I. scapularis genome where it is ~12.7% (28/233) of the degradome as opposed to ~22-48% content in other blood feeding arthropods, Pediculus humanus humanus, Anopheles gambiae, Aedes Aegypti and Culex pipiens quinquefasciatus is notable. The data is presented as a one-stop index of proteases encoded by the I. scapularis genome.     

Characterization of senescence-associated proteases in postharvest broccoli florets.

We characterized the senescence-associated proteases of postharvest broccoli (Brassica oleracea L. var Green King) florets, using class-specific protease inhibitors and gelatin-polyacrylamide gel electrophoresis. Different classes of senescence-associated proteases in broccoli florets were partially characterized for the first time. Protease activity of broccoli florets was depressed by all the inhibitors and showed different inhibition curves during postharvest. The hydrolytic activity of metalloprotease (EC 3.4.24. - ) and serine protease (EC 3.4.21. - ) reached a maximum, 1 day after harvest (DAH), then decreased, while the hydrolytic activity of cysteine protease (EC 3.4.22. - ) and aspartic protease (EC 3.4.23. - ) increased throughout the postharvest senescence based on the calculated inhibition percentage of protease activity. The senescence-associated proteases were separated into seven endoprotease (EP) groups by gelatin-polyacryamide gel electrophoresis and classified into EP1 (metalloprotease), EP2 (metalloprotease and cysteine protease), EP3 (serine protease and aspartic protease), EP4, EP5, EP7 (cysteine protease), and EP6 (serine protease) based on the sensitivity of class-specific protease inhibitors. The proteases EP2, EP3, and EP4 were present throughout the postharvest stages. EP3 was the major EP at all times during senescence; EP4 intensity of activity increased after 2 DAH; EP6 and EP7 clearly increased after 4 DAH. Our results suggest that serine protease activity contributes to early stage (0-1 DAH) and late stage (4-5 DAH) of senescence; metalloprotease activity was involved in the early and intermediate stages (0-3 DAH) of senescence; and cysteine protease and aspartic protease activities participated in the whole process of broccoli senescence.     

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.     

Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage.     

Detection of an Intracellular Protease Inhibitor in Archaea

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a ∼90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea. Received: 4 May 2002 / Accepted: 10 July 2002     

Conformational homogeneity in molecular recognition by proteolytic enzymes

Crystal structures for several hundred protease-inhibitor complexes have been analysed and their superimpositions have been used to demonstrate a universal relationship between inhibitor/substrate conformation and molecular recognition by all aspartic, serine, cysteine and metallo proteases. Proteases universally recognize an extended beta strand conformation in all their peptidic (and non-peptidic) inhibitors and substrate analogues without significant exceptions. This conformational homogeneity is illustrated here for a subset of 180 protease-inhibitor structures which are displayed as (a) structural overlays of multiple inhibitors for each of eight aspartic, eight serine, six metallo and five cysteine proteases; (b) single inhibitors each bound to different proteases; and (c) Ramachandran plots of peptide or pseudo-peptide dihedral angle pairs which demonstrate beta strands (Phi -54 degrees to -173 degrees, Psi 24 degrees to 174 degrees ) like those normally found paired in proteins as beta sheets. However, unlike beta sheets, alpha and 3(10) helices, beta and gamma turns, where the folded main chain amide components are intramolecularly hydrogen bonded and thus unavailable for interaction with proteins, an inhibitor/substrate in an isolated beta strand conformation provides maximum exposure of its hydrogen bonding donors/acceptors and side chain components to a putative protease receptor. This analysis highlights the advantages of a strand conformation over other elements of secondary structure for protease recognition and may lead to generic strategies for inhibitor design.     

Characterization and distribution of digestive proteases of the stalk corn borer,Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae)

Larval midgut extracts from the noctuid Sesamia nonagrioides Lef. were assayed for protease activity. Total proteolytic activity, as measured by azocasein hydrolysis, showed a pH optimum in the range 10.0 to 11.5, suggesting a digestive system based largely on serine-like proteases. The ability of midgut extracts to hydrolyze specific synthetic substrates, the elucidation of the pH at which maximal hydrolysis occurs, and their sensitivity to protease inhibitors confirmed the presence of the serine endoproteases: trypsin, chymotrypsin, and elastase; and the exopeptidases: carboxypeptidase A, carboxypeptidase B, and leucine aminopeptidase. The distribution of these digestive proteases along the gut sections and among the different midgut regions was examined. All types of endoproteases and exopeptidases were mainly located in the midgut, with less than 5% of the activity in the foregut and hindgut. When the two halves of the midgut were compared, all proteolytic activities were higher in the anterior portion of the midgut. Trypsin, chymotrypsin, elastase, and carboxypeptidase B activities were mainly located in the endoperitrophic space of the midgut, with some activity in the ectoperitrophic space, whereas aminopeptidase and carboxypeptidase A activities were preferentially located in the midgut epithelium. © 1996 Wiley-Liss, Inc.     

Processing of chicken progastrin at post-Phe bonds by an aspartyl protease

Prohormones mature to biologically active peptide hormones through posttranslational modifications, which include endoproteolytic cleavages. Cleavages at mono- and dibasic sites are well characterized, and several of the responsible prohormone convertases have been identified. There is, however, evidence that endoproteolytic maturation occurs also at other sites. Among these, post-Phe cleavage occurs in the maturation of chicken progastrin, where the processing to gastrin-30 has been examined in detail. In this study we have characterized an endoprotease of the aspartic acid protease family in chicken and human tissue capable of cleaving at the Phe site. Enzymatic activity was monitored by radioimmunoassays using antibodies specific for the N- and C-termini exposed after cleavage. Analysis showed that only pepstatin, a specific inhibitor of aspartic proteases, inhibited the enzyme. The pH optimum of the enzyme ranged from pH 2 to pH 5. Amino acid substitution from Phe to Ala in the substrate completely abolished enzyme activity. The endoproteolytic activity was identified in chicken antrum and pectoral muscle as well as human cardiac and prostate extracts, suggesting that the enzyme has widespread biological functions. Experiments using recombinant cathepsin D and E indicated that neither is responsible for the endoproteolytic cleavage of chicken progastrin at post-Phe bonds.     

Microbial keratinases and their prospective applications: an overview

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.     

Changes in protein fractions and leucine-[14C] incorporation during Sorghum grain development

Incorporation of leucine and changes in different protein fractions have been studied during Sorghum grain development. Most of the label from the injected leucine-[¹⁴C] was found in glutelin and residue fraction towards later stages of maturity. The label in albumin, globulin and prolamin decreased with a concomitant increase in label in glutelin and residue proteins. The concentration of lysine, aspartic acid and glycine decreased while that of leucine, proline, alanine, tyrosine, phenylalanine, and cystine increased during grain development. Increase in serine, methionine, valine and isoleucine was only marginal. The proportion of glutamic acid was high at all stages of grain development. Glutelin fraction resolved into two peaks on gel chromatography, only one of which with higher MW was labelled, while in albumin both the peaks were found to be labelled. Tannin content also increased during grain development.     

Endoproteolytic activities in pea roots inoculated with the arbuscular mycorrhizal fungus Glomus mosseae and/or Aphanomyces euteiches in relation to bioprotection

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches . First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches , and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae . By contrast, in roots inoculated with A. euteiches , a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as 'trypsin-like' serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.     

Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa

The profile of sedimentation on a 4-20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA. Their activity was maximal at pH 8.0 and increased in the presence of Ca(2+) ions. Both enzymes were inhibited by the chelating agent 1,10-phenanthroline but their thermal inactivation kinetics were different. A second protease peak was observed at 6.8 S and corresponded to a metallo-endoprotease that hydrolyzed with a maximal activity at pH 8.0 only the amide bonds of peptide substrates having a threonine residue at the P1' position. A last protease peak identified at 9.0 S contained a protease that preferentially acted on tripeptides, such as Val-Pro-Leu (diprotin B) and Thr-Val-Leu, releasing the C-terminal residue. Unlike the proteases identified in the two other peaks, its activity was maximal at acid pH (5.0) and was inhibited by the serine protease inhibitors. Together these results show the potential of A. ventricosa as a source of specific proteases.