scholarly journals McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Bence Hajdusits ◽  
Marcin J Suskiewicz ◽  
Nikolas Hundt ◽  
Anton Meinhart ◽  
Robert Kurzbauer ◽  
...  
Keyword(s):  
Active Sites ◽  
Protein Quality ◽  
Arginine Kinase ◽  
Structural Data ◽  
Bacterial Degradation ◽  
Dependent Manner ◽  
Unfolded Proteins ◽  
Gram Positive Bacteria ◽  
Bacterial Proteins

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labelling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labelling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, contrary to these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher-order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

scholarly journals McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation

2020 ◽  
Author(s):  
Bence Hajdusits ◽  
Marcin J. Suskiewicz ◽  
Nikolas Hundt ◽  
Anton Meinhart ◽  
Robert Kurzbauer ◽  
...  
Keyword(s):  
Active Sites ◽  
Protein Quality ◽  
Arginine Kinase ◽  
Structural Data ◽  
Bacterial Degradation ◽  
Dependent Manner ◽  
Unfolded Proteins ◽  
Gram Positive Bacteria ◽  
Bacterial Proteins

AbstractIn Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labelling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labelling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The “closed” octamer interconverts with other oligomers in a phosphorylation-dependent manner and, contrary to these “open” forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher-order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

scholarly journals PapRIV, a BV-2 microglial cell activating quorum sensing peptide

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yorick Janssens ◽  
Nathan Debunne ◽  
Anton De Spiegeleer ◽  
Evelien Wynendaele ◽  
Marta Planas ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Model ◽  
Dependent Manner ◽  
Communication Signals ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

scholarly journals Protection from Lethal Gram-Positive Infection by Macrophage Scavenger Receptor–Dependent Phagocytosis

2000 ◽  
Vol 191 (1) ◽  
pp. 147-156 ◽  
Author(s):  
Christian A. Thomas ◽  
Yongmei Li ◽  
Tatsuhiko Kodama ◽  
Hiroshi Suzuki ◽  
Samuel C. Silverstein ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Critical Role ◽  
Scavenger Receptor ◽  
Lipoteichoic Acid ◽  
Peritoneal Macrophages ◽  
Type I ◽  
Dependent Manner ◽  
Gram Positive ◽  
Gram Positive Bacteria

Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against and recovery from gram-positive infections. Inborn and acquired defects in opsonin generation and/or recognition by phagocytes are associated with an increased susceptibility to bacterial infections. In contrast, the physiological significance of opsonin-independent phagocytosis is unknown. Type I and II class A scavenger receptors (SR-AI/II) recognize a variety of polyanions including bacterial cell wall products such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting a role for SR-AI/II in innate immunity to bacterial infections. Here, we show that SR-AI/II–deficient mice (MSR-A−/−) are more susceptible to intraperitoneal infection with a prototypic gram-positive pathogen, Staphylococcus aureus, than MSR-A+/+ control mice. MSR-A−/− mice display an impaired ability to clear bacteria from the site of infection despite normal killing of S. aureus by neutrophils and die as a result of disseminated infection. Opsonin-independent phagocytosis of gram-positive bacteria by MSR-A−/− macrophages is significantly decreased although their phagocytic machinery is intact. Peritoneal macrophages from control mice phagocytose a variety of gram-positive bacteria in an SR-AI/II–dependent manner. Our findings demonstrate that SR-AI/II mediate opsonin-independent phagocytosis of gram-positive bacteria, and provide the first evidence that opsonin-independent phagocytosis plays a critical role in host defense against bacterial infections in vivo.

scholarly journals The novel chaperone protein SRCP1 reduces insoluble SOD1 protein in vivo without extending ALS mouse lifespan

2020 ◽  
Author(s):  
Ian W. Luecke ◽  
Gloria Lin ◽  
Stephanie Santarriaga ◽  
K. Matthew Scaglione ◽  
Allison D. Ebert
Keyword(s):  
Protein Aggregation ◽  
Motor Neurons ◽  
Protein Misfolding ◽  
Protein Quality ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Insoluble Protein ◽  
Chaperone Protein

AbstractProtein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington’s disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1 G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

scholarly journals Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Ulf Gerth ◽  
Eleonora Krieger ◽  
Daniela Zühlke ◽  
Alexander Reder ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Arginine Kinase ◽  
Adaptor Protein ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Wild Type ◽  
Recognition Mechanism ◽  
Content Type ◽  
In The Wild

ABSTRACT Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

scholarly journals ClpXP Degrades SsrA-Tagged Proteins in Streptococcus pneumoniae

2009 ◽  
Vol 191 (8) ◽  
pp. 2894-2898 ◽  
Author(s):  
Sarita Ahlawat ◽  
Donald A. Morrison
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Protein Quality ◽  
Adaptor Protein ◽  
Translation Process ◽  
E Coli ◽  
Bacterial Proteins ◽  
Rare Codons

ABSTRACT Bacterial proteins that are abnormally truncated due to incomplete mRNA or the presence of rare codons are extended by an SsrA tag during ribosome rescue in a trans-translation process important for maintaining protein quality. In Escherichia coli, the SsrA-tagged proteins become the target of the Tsp, Lon, FtsH, ClpXP, and ClpAP proteases. Here we show that degradation of model SsrA-tagged proteins in Streptococcus pneumoniae depends primarily or exclusively on ClpXP in vivo. In addition, we show the E. coli SsrA tag is also a target of S. pneumoniae ClpXP in vivo, even though the N-terminal portions of the tags differ significantly between the two species, suggesting there may be no adaptor protein for SsrA in S. pneumoniae.

scholarly journals Bivalent antibody pliers inhibit β-tryptase by an allosteric mechanism dependent on the IgG hinge

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Henry R. Maun ◽  
Rajesh Vij ◽  
Benjamin T. Walters ◽  
Ashley Morando ◽  
Janet K. Jackman ◽  
...  
Keyword(s):  
Active Sites ◽  
Inflammatory Responses ◽  
Structural Data ◽  
Allosteric Modulation ◽  
Antibody Engineering ◽  
Dependent Manner ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Important Mediator ◽  
Allosteric Mechanism

AbstractHuman β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.

scholarly journals Viral vector gene delivery of the novel chaperone protein SRCP1 to modify insoluble protein in in vitro and in vivo models of ALS

Gene Therapy ◽  
2021 ◽  
Author(s):  
Ian W. Luecke ◽  
Gloria Lin ◽  
Stephanie Santarriaga ◽  
K. Matthew Scaglione ◽  
Allison D. Ebert
Keyword(s):  
Protein Aggregation ◽  
Protein Quality ◽  
Viral Vector ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Insoluble Protein ◽  
In Vivo Models ◽  
Chaperone Protein

AbstractProtein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington’s disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1-G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

scholarly journals Myeloperoxidase and Eosinophil Peroxidase Inhibit the in vitro Endotoxin Activities of Lipopolysaccharide (LPS) and Lipid A and Increase Survival in an in vivo Mouse LPS LD90 model

2018 ◽  
Author(s):  
Robert C. Allen ◽  
Mary L. Henery ◽  
John C. Allen ◽  
Roger J. Hawks ◽  
Jackson T. Stephens
Keyword(s):  
Lipid A ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Gram Positive Bacteria ◽  
Enzymatic Function ◽  
Eosinophil Peroxidase ◽  
Kaplan Meier ◽  
Endotoxin Activity

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic leukocyte haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds and kills specific Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. Endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic Lipid A component, is a characteristic of all GNB. The possibility that haloperoxidases bind to and inhibit the endotoxin of GBN was considered and tested by contacting MPO and EPO with LPS and Lipid A and measuring for inhibition of endotoxin activity using either the in vitro gel or chromogenic Limulus amebocyte lysate (LAL) assays. Contacting MPO and EPO with LPS purified from Escherichia coli O55:B5 and with diphosphoryl Lipid A purified from E. coli F583 inhibited their endotoxin activities in proportion to the natural log of the MPO or EPO concentration. Although MPO is less cationic than EPO, MPO consistently demonstrated inhibition of endotoxin activity that is about threefold superior to EPO. Haloperoxidase enzymatic activity was not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. MPO and EPO inhibition of LPS endotoxin activity was also measured using a 90% lethal dose (LD90) mouse model studied over a five-day period. Based on Kaplan Meier survival analysis, MPO significantly increased mouse survival in a dose-dependent manner. EPO was less effective. In conclusion, contacting MPO and EPO with LPS and Lipid A inhibits in vitro endotoxin activities, but inhibition is independent of haloperoxidase enzymatic function. MPO significantly increases mouse survival against LPS in an in vivo LD90 endotoxin model.

Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

1996 ◽  
Vol 76 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Yasuto Sasaki ◽  
Junji Seki ◽  
John C Giddings ◽  
Junichiro Yamamoto
Keyword(s):  
Blood Flow ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Red Cell ◽  
Cell Velocity ◽  
Red Cell Velocity ◽  
The Mean ◽  
Wall Shear ◽  
Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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