scholarly journals Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Vol 117 (45) ◽  
pp. 27989-27996
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Wild Type ◽  
Protease Domain ◽  
Loop Region ◽  
Assembly Pathway

Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

scholarly journals Reversible auto-inhibitory regulation of Escherichia coli metallopeptidase BepA for selective β-barrel protein degradation

2020 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Ryoji Miyazaki ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Protease Activity ◽  
Underlying Mechanism ◽  
Zinc Ion ◽  
Loop Region ◽  
Assembly Pathway ◽  
Inhibitory Regulation

AbstractEscherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD, but the underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features, in particular the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has a potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of the BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of the protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

In Vitro and in Vivo Neutralization of Murine Activated Protein C

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3364-3364
Author(s):  
Laurent Burnier ◽  
Jose A. Fernandez ◽  
John H. Griffin
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Activity Level ◽  
Protective Effects ◽  
Wild Type ◽  
Amidolytic Activity ◽  
Endogenous Protein

Abstract Abstract 3364 Activated Protein C (APC) is a circulating serine protease with two major roles to maintain homeostasis. APC acts via multiple receptors, including protease-activated receptor 1, to exert anti-apoptotic and vascular integrity protective effects. A number of protective effects of pharmacologic APC are reported in the literature, with beneficial effects in kidney, brain and irradiation-induced pathologies. The functional protections of the endogenous protein C systems are challenging to study. A better understanding of its mechanisms at different cellular levels and in different tissues is needed to enable evaluation of its further usage in humans. To that end, new tools should be considered to increase our knowledge. To help evaluate the endogenous murine protein C system and to be able to neutralize pharmacologic APC, we have made and characterized a novel rat monoclonal anti-mouse protein C antibody, SPC-54, that almost completely ablates in vitro and in vivo APC activity. In solid phase binding assays, the Kd of SPC-54 for APC was about 8 nM. In biochemical assays, SPC-54 inhibited amidolytic activity of wild-type murine APC by > 95%. SPC-54 was similarly a potent inhibitor (> 90%) of the amidolytic activity of the 5A-APC mutant. IC50 value for wild-type APC and the 5A-APC mutant were comparable. SPC-54 was pre-incubated with APC, followed by the addition of a 20 fold molar excess of biotinylated FPR-chloromethylketone, quantification of biotinylation of APC was readily made by SDS-PAGE and Western blots using infrared-coupled streptavidin. SPC-54 blocked successfully active site titration of APC using this biotinylated active site titrant. These and other experiments suggest that the SPC-54 epitope is located in the vicinity of the active site, such that it blocks different small substrates from reaching the active site. When we performed thrombin generation assays using mouse platelet-poor plasma to check whether SPC-54 was a potent blocker of APC activity in plasma, we showed that SPC-54 neutralized almost completely exogenous APC anticoagulant activity in a dose-dependent manner. Using native polyacrylamide gel migration, Western immunoblotting and immuno-precipitation with protein G-agarose, we confirmed that SPC-54 was bound to protein C in plasma after infusing mice with SPC-54 (10 mg/kg). Moreover, using a modified ELISA that is capable to quantify the pool of activatable protein C, the plasma protein C activity level was considerably decreased (> 80%) in mice after a single injection of SPC-54 (10 mg/kg), and that this effect of neutralizing circulating protein C was sustained for at least 7 days. For in vivo proof of concept, we performed murine tissue factor-induced thromboembolism experiments. Results showed a severe decrease in survival of mice that were pre-infused with SPC-54 when compared to control (survival time of 7 min vs. 42.5 min respectively, P = 0.0016). Moreover, blood perfusion in lungs of mice infused with SPC-54 (10 mg/kg) was dramatically impaired (decrease of 54%, P < 0.0001) as revealed by infrared quantification of Evans Blue dye as marker of vascular perfusion. We also used endotoxemia murine models to assess effects of SPC-54. SPC-54 decreased survival after endotoxin challenge (25 mg/kg, LD50 dose) in mice infused with SPC-54 (10 mg/kg) at 7 hours after LPS. Mortality was 100% after 36 h in the SPC-54 group, whereas controls, which received either boiled SPC-54 antibodies or PBS vehicle, showed a mortality of about 50% (P < 0.001). In summary, SPC-54 is a potent rat monoclonal antibody that neutralizes murine APC activities in vitro and in vivo. Its characteristic ability to dampen the endogenous protein C/APC system is of value to understand better the role of the endogenous protein C system in murine injury models and also to neutralize pharmacologic murine APC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mechanisms of p34cdc2 regulation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685 ◽  
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

Mechanisms of p34cdc2 regulation

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

2006 ◽  
Vol 74 (11) ◽  
pp. 6124-6134 ◽  
Author(s):  
Nathalie M. Maroncle ◽  
Kelsey E. Sivick ◽  
Rebecca Brady ◽  
Faye-Ellen Stokes ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Active Site ◽  
Protease Activity ◽  
Host Cells ◽  
Single Mutation ◽  
Wild Type ◽  
Passenger Domain ◽  
Hek 293

ABSTRACT The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

scholarly journals DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

2021 ◽  
Author(s):  
Jie Shen ◽  
Shengjie Feng ◽  
Jiao Deng ◽  
Qingwen Huang ◽  
Dayong Zhao ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Animal Experiments ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Wild Type ◽  
Vitro Assay ◽  
Cell Invasiveness ◽  
The Stability

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

scholarly journals Mutations that improve the binding of yeast FLP recombinase to its substrate.

Genetics ◽  
1988 ◽  
Vol 118 (3) ◽  
pp. 393-400
Author(s):  
B Lebreton ◽  
P V Prasad ◽  
M Jayaram ◽  
P Youderian
Keyword(s):  
Active Site ◽  
Promoter Region ◽  
Protein A ◽  
Wild Type ◽  
Flp Recombinase ◽  
Active Site Tyrosine ◽  
Synthetic Target ◽  
Better Than

Abstract When yeast FLP recombinase is expressed from the phage lambda PR promoter in a Salmonella host, it cannot efficiently repress an operon controlled by an operator/promoter region that includes a synthetic, target FLP site. On the basis of this phenotype, we have identified four mutant FLP proteins that function as more efficient repressors of such an operon. At least two of these mutant FLP proteins bind better to the FLP site in vivo and in vitro. One mutant changes the presumed active site tyrosine residue of FLP protein to phenylalanine, is blocked in recombination, and binds the FLP site about five-fold better than the wild-type protein. A second mutant protein that functions as a more efficient repressor retains catalytic activity. We conclude that the eukaryotic yeast FLP recombinase, when expressed in a heterologous prokaryotic host, can function as a repressor, and that mutant FLP proteins that bind DNA more tightly may be selected as more efficient repressors.

scholarly journals Phenotypic Characterization of Resistant Val36 Variants of Hepatitis C Virus NS3-4A Serine Protease

2007 ◽  
Vol 52 (1) ◽  
pp. 110-120 ◽  
Author(s):  
Yi Zhou ◽  
Doug J. Bartels ◽  
Brian L. Hanzelka ◽  
Ute Müh ◽  
Yunyi Wei ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
Phenotypic Characterization ◽  
Replication Capacity ◽  
Wild Type ◽  
Protease Domain

ABSTRACT In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had ∼20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-Å resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

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