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

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34 + /CD45 + monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34 + /CD45 + cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010301117 ◽

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.

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In Vitro and in Vivo Neutralization of Murine Activated Protein C

Blood ◽

10.1182/blood.v120.21.3364.3364 ◽

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.

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Mechanisms of p34cdc2 regulation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1675 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1675-1685 ◽

Cited By ~ 113

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.

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Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival

Infection and Immunity ◽

10.1128/iai.00088-09 ◽

2009 ◽

Vol 77 (7) ◽

pp. 2783-2794 ◽

Cited By ~ 40

Author(s):

Scarlett Geunes-Boyer ◽

Timothy N. Oliver ◽

Guilhem Janbon ◽

Jennifer K. Lodge ◽

Joseph Heitman ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Defense Mechanisms ◽

Protein A ◽

Surfactant Protein ◽

Immune Defense ◽

Surfactant Protein D ◽

Wild Type ◽

Phagocytic Cells

ABSTRACT Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Δ mutant hypocapsular strain are assessed. SP-D binding to cap59Δ mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Δ cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D−/− mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Δ or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D−/− mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy.

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

Nucleic Acids Research ◽

10.1093/nar/gkz1152 ◽

2019 ◽

Vol 48 (2) ◽

pp. 847-861 ◽

Cited By ~ 2

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.

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Mechanisms of p34cdc2 regulation

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1675-1685.1993 ◽

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.

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Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

Infection and Immunity ◽

10.1128/iai.66.5.2180-2185.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2180-2185 ◽

Cited By ~ 35

Author(s):

Karsten R. O. Hazlett ◽

Suzanne M. Michalek ◽

Jeffrey A. Banas

Keyword(s):

Streptococcus Mutans ◽

Mutant Strain ◽

Rat Model ◽

Binding Protein ◽

Protein A ◽

Wild Type ◽

Mutant Strains

ABSTRACT Glucan-binding protein A (GbpA) of Streptococcus mutanshas been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenicgbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutanswas grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. ThegbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB andgtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

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Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo

Journal of Virology ◽

10.1128/jvi.00280-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 6009-6016 ◽

Cited By ~ 48

Author(s):

Sindy Böttcher ◽

Christina Maresch ◽

Harald Granzow ◽

Barbara G. Klupp ◽

Jens P. Teifke ◽

...

Keyword(s):

Active Site ◽

Pseudorabies Virus ◽

Wild Type Virus ◽

Tegument Protein ◽

Wild Type ◽

Active Site Cysteine ◽

Specific Protease ◽

Ubiquitin Specific Protease

ABSTRACT Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C26S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C26S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.

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Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2267 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2267-2280 ◽

Cited By ~ 25

Author(s):

Kara Dolinski ◽

Christian Scholz ◽

R. Scott Muir ◽

Sabine Rospert ◽

Franz X. Schmid ◽

...

Keyword(s):

Active Site ◽

Active Sites ◽

In Vitro Assay ◽

Biologically Active ◽

Wild Type ◽

Prolyl Isomerase ◽

Vitro Assay ◽

Isomerase Activity

Cyclophilin and FK506 binding protein (FKBP) acceleratecis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

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