scholarly journals The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers

1999 ◽  
Vol 345 (1) ◽  
pp. 129-138 ◽  
Author(s):  
Magali PARIAT ◽  
Catherine SALVAT ◽  
Magali BÉBIEN ◽  
Frédérique BROCKLY ◽  
Eléna ALTIERI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Structural Integrity ◽  
Quaternary Structure ◽  
Cysteine Proteases ◽  
Site Directed Mutagenesis ◽  
Fos Protein ◽  
Highly Sensitive ◽  
Strict Specificity ◽  
Rate Limiting

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to Pʹ2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

2020 ◽  
pp. jbc.RA120.015524
Author(s):  
Alexandra P Breslawec ◽  
Shaochi Wang ◽  
Crystal Li ◽  
Myles B Poulin
Keyword(s):  
Amino Acids ◽  
Bacterial Infections ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Binding Surface ◽  
Activity Measurements ◽  
Rate Of Hydrolysis ◽  
Biofilm Dispersal ◽  
Hydrolysis Of

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the –1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

scholarly journals Production and Physicochemical Characterization of Gelatin and Collagen Hydrolysates from Turbot Skin Waste Generated by Aquaculture Activities

Marine Drugs ◽  
2021 ◽  
Vol 19 (9) ◽  
pp. 491
Author(s):  
Jesus Valcarcel ◽  
Javier Fraguas ◽  
Carolina Hermida-Merino ◽  
Daniel Hermida-Merino ◽  
Manuel M. Piñeiro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Structural Integrity ◽  
Physicochemical Characterization ◽  
Essential Amino Acids ◽  
Storage And Loss Moduli ◽  
Food And Feed ◽  
Collagen Hydrolysates ◽  
First Time ◽  
By Products

Rising trends in fish filleting are increasing the amount of processing by-products, such as skins of turbot, a flatfish of high commercial value. In line with circular economy principles, we propose the valorization of turbot skins through a two-step process: initial gelatin extraction described for the first time in turbot, followed by hydrolysis of the remaining solids to produce collagen hydrolysates. We assayed several methods for gelatin extraction, finding differences in gelatin properties depending on chemical treatment and temperature. Of all methods, the application of NaOH, sulfuric, and citric acids at 22 °C results in the highest gel strength (177 g), storage and loss moduli, and gel stability. We found no relation between mechanical properties and content of pyrrolidine amino acids, but the best performing gelatin displays higher structural integrity, with less than 30% of the material below 100 kDa. Collagen hydrolysis was more efficient with papain than alcalase, leading to a greater reduction in Mw of the hydrolysates, which contain a higher proportion of essential amino acids than gelatin and show high in vitro anti-hypertensive activity. These results highlight the suitability of turbot skin by-products as a source of gelatin and the potential of collagen hydrolysates as a functional food and feed ingredient.

scholarly journals The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation

2002 ◽  
Vol 76 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Jeffery Tuckis ◽  
Sherin Smallwood ◽  
Joyce A. Feller ◽  
Sue A. Moyer
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Sendai Virus ◽  
Intact Cell ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Multiple Functions

ABSTRACT The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP0 binding and abolished the reconstitution of replication from separate P-L and NP0-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP0-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.

scholarly journals Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001468
Author(s):  
Gabriel Piedrafita ◽  
Sreejith J. Varma ◽  
Cecilia Castro ◽  
Christoph Messner ◽  
Lukasz Szyrwiel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metabolic Network ◽  
Reaction Network ◽  
High Specificity ◽  
Pentose Phosphate ◽  
Evolutionary Origins ◽  
Metal Catalyzed ◽  
Rate Limiting ◽  
Nonenzymatic Reactions

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

1998 ◽  
Vol 64 (6) ◽  
pp. 1991-1996 ◽  
Author(s):  
Benedicte Flambard ◽  
Sandra Helinck ◽  
Jean Richard ◽  
Vincent Juillard
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Essential Amino Acids ◽  
Genetically Engineered ◽  
Amino Acid Requirements ◽  
Rate Limiting

ABSTRACT The ability of caseins to fulfill the amino acid requirements ofLactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

Processing and trafficking of cysteine proteases in Leishmania mexicana

2000 ◽  
Vol 113 (22) ◽  
pp. 4035-4041 ◽  
Author(s):  
D.R. Brooks ◽  
L. Tetley ◽  
G.H. Coombs ◽  
J.C. Mottram
Keyword(s):  
Cysteine Protease ◽  
Mammalian Cells ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Flagellar Pocket ◽  
Leishmania Mexicana

Removal of the pro-domain of a cysteine protease is essential for activation of the enzyme. We have engineered a cysteine protease (CPB2.8) of the protozoan parasite Leishmania mexicana by site-directed mutagenesis to remove the active site cysteine (to produce CPB(C25G)). When CPB(C25G) was expressed in a L. mexicana mutant lacking all CPB genes, the inactive pro-enzyme was processed to the mature protein and trafficked to the lysosome. These results show that auto-activation is not required for correct processing of CPB in vivo. When CPB(C25G) was expressed in a L. mexicana mutant lacking both CPA and CPB genes, the majority of the pro-enzyme remained unprocessed and accumulated in the flagellar pocket. These data reveal that CPA can directly or indirectly process CPB(C25G) and suggest that cysteine proteases are targeted to lysosomes via the flagellar pocket. Moreover, they show that another protease can process CPB in the absence of either CPA or CPB, albeit less efficiently. Abolition of the glycosylation site in the mature domain of CPB did not affect enzyme processing, targeting or in vitro activity towards gelatin. This indicates that glycosylation is not required for trafficking. Together these findings provide evidence that the major route of trafficking of Leishmania cysteine proteases to lysosomes is via the flagellar pocket and therefore differs significantly from cysteine protease trafficking in mammalian cells.

scholarly journals Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 642
Author(s):  
David Peterhoff ◽  
Stefanie Thalhauser ◽  
Jan M. Sobczak ◽  
Mona O. Mohsen ◽  
Christoph Voigt ◽  
...  
Keyword(s):  
Colloidal Stability ◽  
Structural Integrity ◽  
Quaternary Structure ◽  
Silica Nanoparticle ◽  
Neutralizing Antibody ◽  
Surface Attachment ◽  
Nanoparticle Delivery ◽  
Clade C ◽  
Hiv 1

The delivery of HIV-1 envelope (Env) trimer-based immunogens on the surface of nanoparticles holds promise to promote immunogenicity with the aim of inducing a potent, durable and broad neutralizing antibody (bnAb) response. Towards that goal, we examined the covalent conjugation of Env to 100 nm and 200 nm silica nanoparticles (SiNPs) to optimize conjugation density and attachment stability. Env was redesigned to enable site-specific cysteine-mediated covalent conjugation while maintaining its structural integrity and antigenicity. Env was anchored to different sized SiNPs with a calculated spacing of 15 nm between adjacent trimers. Both particle sizes exhibited high in vitro stability over a seven-day period. After attachment, 100 nm particles showed better colloidal stability compared to 200 nm particles. Importantly, the antigenic profile of Env was not impaired by surface attachment, indicating that the quaternary structure was maintained. In vitro Env uptake by dendritic cells was significantly enhanced when Env was delivered on the surface of nanoparticles compared to soluble Env. Furthermore, multivalent Env displayed efficiently activated B cells even at Env concentrations in the low nanomolar range. In mice, antibody responses to nanoparticle-coupled Env were stronger compared to the free protein and had equivalent effects at lower doses and without adjuvant.

scholarly journals Identification of Two Mutations (F758W and F758Y) in the N -methyl-D-aspartate Receptor Glycine-binding Site that Selectively Prevent Competitive Inhibition by Xenon without Affecting Glycine Binding

2012 ◽  
Vol 117 (1) ◽  
pp. 38-47 ◽  
Author(s):  
Scott P. Armstrong ◽  
Paul J. Banks ◽  
Thomas J. W. McKitrick ◽  
Catharine H. Geldart ◽  
Christopher J. Edge ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nmda Receptor ◽  
Binding Site ◽  
Competitive Inhibition ◽  
Site Directed Mutagenesis ◽  
Glycine Site ◽  
General Anesthetic ◽  
Hek 293

Background Xenon is a general anesthetic with neuroprotective properties. Xenon inhibition at the glycine-binding site of the N-Methyl-D-aspartate (NMDA) receptor mediates xenon neuroprotection against ischemic injury in vitro. Here we identify specific amino acids important for xenon binding to the NMDA receptor, with the aim of finding silent mutations that eliminate xenon binding but leave normal receptor function intact. Methods Site-directed mutagenesis was used to mutate specific amino-acids in the GluN1 subunit of rat NMDA receptors. Mutant GluN1/GluN2A receptors were expressed in HEK 293 cells and were assessed functionally using patch-clamp electrophysiology. The responses of the mutant receptors to glycine and anesthetics were determined. Results Mutation of phenylalanine 758 to an aromatic tryptophan or tyrosine left glycine affinity unchanged, but eliminated xenon binding without affecting the binding of sevoflurane or isoflurane. Conclusions These findings confirm xenon binds to the glycine site of the GluN1 subunit of the NMDA receptor and indicate that interactions between xenon and the aromatic ring of the phenylalanine 758 residue are important for xenon binding. Our most important finding is that we have identified two mutations, F758W and F758Y, that eliminate xenon binding to the NMDA receptor glycine site without changing the glycine affinity of the receptor or the binding of volatile anesthetics. The identification of these selective mutations will allow knock-in animals to be used to dissect the mechanism(s) of xenon's neuroprotective and anesthetic properties in vivo.

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