scholarly journals The Serine Protease HhoA from Synechocystis sp. Strain PCC 6803: Substrate Specificity and Formation of a Hexameric Complex Are Regulated by the PDZ Domain

2007 ◽  
Vol 189 (18) ◽  
pp. 6611-6618 ◽  
Author(s):  
Pitter F. Huesgen ◽  
Philipp Scholz ◽  
Iwona Adamska
Keyword(s):  
Membrane Proteins ◽  
Substrate Specificity ◽  
Proteolytic Activity ◽  
Protease Activity ◽  
Elevated Temperatures ◽  
Critical Role ◽  
Pdz Domain ◽  
General Role ◽  
Pcc 6803

ABSTRACT Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg2+ or Ca2+. We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled β-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a ΔhhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.

scholarly journals FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Proteolytic Activity ◽  
Guinea Pigs ◽  
Protease Activity ◽  
Amino Acid Esters ◽  
Protein Substrates ◽  
Protease Activation ◽  
Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

scholarly journals Suppressor Mutations indegSOvercome the Acute Temperature-Sensitive Phenotype of ΔdegPand ΔdegPΔtol-palMutants ofEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Brea Kern ◽  
Owen P. Leiser ◽  
Rajeev Misra
Keyword(s):  
Protease Activity ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Pdz Domain ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Salt Bridges ◽  
Suppressor Mutations ◽  
Growth Phenotype ◽  
Complex Display

ABSTRACTInEscherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in thedegSgene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS’s protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS’s basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS’s protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolBΔdegPmutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCEThe Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP’s periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.

scholarly journals Effect of different protein sources on protease activity of northern pike, Esox lucius Linneaus 1758, juvenile

2020 ◽  
Vol 37 (2) ◽  
pp. 135-138
Author(s):  
Cemil Kaya GÖKÇEK ◽  
Tamás SZABÓ ◽  
Cüneyt SUZER
Keyword(s):  
Proteolytic Activity ◽  
Protease Activity ◽  
Fish Meal ◽  
Soybean Protein ◽  
Northern Pike ◽  
Inhibitory Effect ◽  
Live Prey ◽  
Protein Sources ◽  
Corn Gluten

The aim of the study is to determine the inhibitory effect of different protein sources on protease activity of Northern pike, E. Lucius, during larval ontogeny. For this purpose, Northern pike were fed from yolk sac absorption until 21 days after hatching (DAH). At that point, larvae were sampled on 7, 14 and 21 DAH days and the activity of enzyme was analyzed in vitro. In the study, two different fish meal, chicken meal, krill meal, corn gluten, soybean protein concentrate, soybean meal and dried distillers grains with solubles were tested. Fish meal-I showed the lowest effect (7.53 %) on 7 DAH larvae. Moreover, chicken meal has the highest inhibitory effect on the proteases in the first week (68.27%). In the following period (DAH 14), although the inhibition ratio dramatically increased in all ingredients, fish meal-I has still the lowest effect on proteolytic activity (55.66%). In the same period, the highest effect was obtained from krill (82.28 %) and chicken meals (86.73 %), respectively. Then, there was no statistical difference between fish meal-I, fish meal-II and corn gluten in the 21 DAH and relatively lower than the others (p>0.05). Additionally, chicken meal again has the highest effect on juveniles with the ratio 89.27 %. As a result, the increase of proteolytic activity was notably increased in 7-14-21 DAH, however, it is concluded that feeding larvae and juveniles with live prey is still suggested to get better result for such a carnivorous species culture.

scholarly journals The vimE Gene Downstream of vimA Is Independently Expressed and Is Involved in Modulating Proteolytic Activity in Porphyromonas gingivalis W83

2004 ◽  
Vol 72 (10) ◽  
pp. 5555-5564 ◽  
Author(s):  
Elaine Vanterpool ◽  
Francis Roy ◽  
Hansel M. Fletcher
Keyword(s):  
Proteolytic Activity ◽  
Porphyromonas Gingivalis ◽  
Protease Activity ◽  
Catalytic Domain ◽  
Immunoblot Analysis ◽  
Extracellular Protein ◽  
Transcriptional Unit ◽  
Wild Type ◽  
Defective Mutant

ABSTRACT Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A unique 1.3-kb open reading frame downstream of the bcp-recA-vimA transcriptional unit was cloned, insertionally inactivated with the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, the growth rate of the mutant strain (designated FLL93) was reduced, and when plated on Brucella blood agar it was nonpigmented and nonhemolytic. Arginine- and lysine-specific gingipain activities were reduced by approximately 90 and 85%, respectively, relative to activities of the parent strain. These activities were unaffected by the culture's growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92, which has increased proteolytic activity in stationary phase. Expression of the rgpA, rgpB, and kgp gingipain genes was unaltered in P. gingivalis FLL93 compared to that of the wild-type strain. Further, in extracellular protein fractions a 64-kDa band was identified that was immunoreactive with the RgpB-specific proenzyme antibodies. Active-site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone or immunoblot analysis showed no detectable protein band representing the gingipain catalytic domain. In vitro protease activity could be slightly induced by a urea denaturation-renaturation cycle in an extracellular protein fraction, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of flanking genes, including recA, vimA, and Pg0792, was unaltered by the mutation. Taken together, these results suggest that the vimA downstream gene, designated vimE (for virulence-modulating gene E), is involved in the regulation of protease activity in P. gingivalis.

scholarly journals Profile of the body surface proteolytic systém in Apis mellifera quee

2011 ◽  
Vol 56 (No. 1) ◽  
pp. 15-22 ◽  
Author(s):  
A.J. Strachecka ◽  
M.M. Gryzińska ◽  
M. Krauze ◽  
K. Grzywnowicz
Keyword(s):  
Protease Inhibitor ◽  
Honey Bee ◽  
Proteolytic Activity ◽  
Body Surface ◽  
Protease Activity ◽  
Serine Proteases ◽  
Developmental Stages ◽  
Cysteine Proteases ◽  
The Body

The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.

scholarly journals The cryo-EM structure of the chloroplast ClpP complex reveals an interaction with the co-chaperonin complex that inhibits ClpP proteolytic activity

2021 ◽  
Author(s):  
Ning Wang ◽  
Yifan Wang ◽  
Qian Zhao ◽  
Xiang Zhang ◽  
Chao Peng ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Control System ◽  
Proteolytic Activity ◽  
Protease Activity ◽  
Protein Quality ◽  
Protein Homeostasis ◽  
Clp Protease ◽  
Protein Quality Control System

Protein homeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. We determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtiiby cryo-EM. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

scholarly journals A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

1997 ◽  
Vol 17 (11) ◽  
pp. 6736-6745 ◽  
Author(s):  
S Kagaya ◽  
C Kitanaka ◽  
K Noguchi ◽  
T Mochizuki ◽  
A Sugiyama ◽  
...  
Keyword(s):  
Protease Activity ◽  
Serine Proteases ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Interleukin 1 ◽  
Critical Role ◽  
Adaptor Proteins ◽  
Cell Lysates

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.

scholarly journals The C-terminal Tail of Presenilin Regulates Omi/HtrA2 Protease Activity

2004 ◽  
Vol 279 (44) ◽  
pp. 45844-45854 ◽  
Author(s):  
Sanjeev Gupta ◽  
Rajesh Singh ◽  
Pinaki Datta ◽  
ZhiJia Zhang ◽  
Christopher Orr ◽  
...  
Keyword(s):  
Cell Death ◽  
Proteolytic Activity ◽  
Protease Activity ◽  
Molecular Mechanisms ◽  
Pdz Domain ◽  
Ectopic Expression ◽  
Amyloid Β ◽  
Peptide Ligand ◽  
Binding Motif ◽  
Dependent Manner

Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease. Presenilins play an important role in amyloid β-precursor processing, NOTCH receptor signaling, and apoptosis. However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood. Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain. We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and β-casein and induces cell death in an Omi/HtrA2-dependent manner. Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death. Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis. This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins.

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 873-881 ◽  
Author(s):  
Claire Lewis ◽  
Henrieta Skovierova ◽  
Gary Rowley ◽  
Bronislava Rezuchova ◽  
Dagmar Homerova ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protease Activity ◽  
Pdz Domain ◽  
Proximal Promoter ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Serovar Typhimurium

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ E (RpoE). S. Typhimurium htrA also possesses a σ E-dependent promoter; however, we found that the absence of σ E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ 70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein

1991 ◽  
Vol 37 (10) ◽  
pp. 758-763 ◽  
Author(s):  
D. D. Rockey ◽  
P. S. D Turaga ◽  
G. D. Wiens ◽  
B. A. Cook ◽  
S. L. Kaattari
Keyword(s):  
Cell Surface ◽  
Proteolytic Activity ◽  
Elevated Temperatures ◽  
Surface Protein ◽  
Major Constituent ◽  
Lauryl Sulfate ◽  
Cell Surface Protein ◽  
Renibacterium Salmoninarum ◽  
Native Form

Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 °C but became readily apparent at 37 °C. Proteinase activity was detected at bacterial physiological temperatures (17 °C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 °C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol). Key words: Renibacterium salmoninarum, proteinase, hemagglutinin, antigen F, bacterial kidney disease.

Sign in / Sign up

Export Citation Format

Share Document