a-pipeline-to-investigate-structures-signaling-pathways-sphingosine-1

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Sichuan University

Preparation, characterization and cellular biocompatibility study of a series of calcium polyphosphate containing 0-100 mol% of Ca2+ replaced by Sr2+ were reported. The osteoblastic ROS17/2.8 cell line was used and seeded on the strontium-doped calcium polyphosphate (SCPP) scaffolds to estimate its optimal dose and to study its potential to support the growth of osteoblastic cells for bone tissue engineering. The effects of SCPP on cells' proliferation and differentiation were evaluated by MTT and ALP activity assay. The results showed that porous SCPP did not exert cytotoxic effect on the cells. In addition, the proliferation and differentiation of the growth of ROS17/2.8 cells on the SCPP containing a low dose of strontium showed a higher level compared to the control, and the SCPP containing 1% strontium was optimal according to the results of MTT and ALP activity assay. The cells on the porous SCPP formed a continuous layer on the outer and inner surface observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The bunchy collagens were excreted from the cells and the calcium granules wrapped by collagens were sedimentated on the surface of cells. The results suggested that the biodegradable SCPP could stimulate the proliferation and differentiation of ROS17/2.8 cells in vitro after addition of proper dose of strontium. The porous SCPP may be a promising material for the bone tissue engineering.

Effect of strontium ions on the growth of ROS17/2.8 cells on porous calcium polyphosphate scaffolds.

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from  (PaAK), a typical α2β2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2β2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.

Mechanistic insights into the allosteric regulation of  aspartate kinase.

Biofilm formation is a critical determinant in the pathopoiesis of  It could significantly increase bacterial resistance to drugs and host defense. Thus, inhibition of biofilm matrix production could be regarded as a promising attempt to prevent colonization of  and the subsequent infection. PpgL, a periplasmic gluconolactonase, has been reported to be involved in  quorum-sensing (QS) system regulation. However, the detailed function and catalysis mechanism remain elusive. Here, the crystal structure of PpgL is described in the current study, along with biochemical analysis, revealing that PpgL is a typical β-propeller enzyme with unique metal-independent lactone hydrolysis activity. Consequently, comparative analysis of seven-bladed propeller lactone-catalyzing enzymes and mutagenesis studies identify the critical sites which contribute to the diverse catalytic and substrate recognition functions. In addition, the reduced biofilm formation and attenuated invasion phenotype resulting from deletion of  confirm the importance of PpgL in  pathogenesis. These results suggest that PpgL is a potential target for developing new agents against the diseases caused by .

Structural and Functional Insights into PpgL, a Metal-Independent β-Propeller Gluconolactonase That Contributes to  Virulence.

MucA and MucB are critical negative modulators of sigma factor AlgU and regulate the mucoid conversion of Pseudomonas aeruginosa. Previous studies have revealed that lipid signals antagonize MucA-MucB binding. Here we report the crystal structure of MucB in complex with the periplasmic domain of MucA and polyethylene glycol (PEG), which unveiled an intermediate state preceding the MucA-MucB dissociation. Based on the biochemical experiments, the aliphatic side chain with a polar group was found to be of primary importance for inducing MucA cleavage. These results provide evidence that the hydrophobic cavity of MucB is a primary site for sensing lipid molecules and illustrates the detailed control of conformational switching within MucA-MucB in response to lipophilic effectors.

Molecular basis of the lipid-induced MucA-MucB dissociation in Pseudomonas aeruginosa.

ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation.

Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria.

Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element.

Glycosylation possess prominent biological and pharmacological significance in natural product and drug candidate synthesis. The glycosyltransferase YjiC, discovered from Bacillus subtilis (Bs-YjiC), shows potential applications in drug development due to its wide substrate spectrums. In order to elucidate its catalytic mechanism, we solved the crystal structure of Bs-YjiC, demonstrating that Bs-YjiC adopts a typical GT-B fold consisting of a flexible N-domain and a relatively rigid C-domain. Structural analysis coupled with site-directed mutagenesis studies revealed that site Ser277 was critical for Nucleoside Diphosphate (NDP) recognition, while Glu317, Gln318, Ser128 and Ser129 were crucial for glycosyl moiety recognition. Our results illustrate the structural basis for acceptor promiscuity in Bs-YjiC and provide a starting point for further protein engineering of Bs-YjiC in industrial and pharmaceutical applications.

Structural and biochemical studies of the glycosyltransferase Bs-YjiC from Bacillus subtilis.

Type II toxin-antitoxin (TA) systems modulate many essential cellular processes in prokaryotic organisms. Recent studies indicate certain type II antitoxins also transcriptionally regulate other genes, besides neutralizing toxin activity. Herein, we investigated the diverse transcriptional repression properties of type II TA antitoxin PaHigA from Pseudomonas aeruginosa. Biochemical and functional analyses showed that PaHigA recognized variable pseudopalindromic DNA sequences and repressed expression of multiple genes. Furthermore, we presented high resolution structures of apo-PaHigA, PaHigA-P and PaHigA-P complex, describing how the rearrangements of the HTH domain accounted for the different DNA-binding patterns among HigA homologues. Moreover, we demonstrated that the N-terminal loop motion of PaHigA was associated with its apo and DNA-bound states, reflecting a switch mechanism regulating HigA antitoxin function. Collectively, this work extends our understanding of how the PaHigB/HigA system regulates multiple metabolic pathways to balance the growth and stress response in P. aeruginosa and could guide further development of anti-TA oriented strategies for pathogen treatment.

Pseudomonas aeruginosa antitoxin HigA functions as a diverse regulatory factor by recognizing specific pseudopalindromic DNA motifs.

AlgW, a membrane-bound periplasmic serine protease belonging to the HtrA protein family, is a key regulator of the regulated intramembrane proteolysis (RIP) pathway and is responsible for transmitting the envelope stress signals in  The AlgW PDZ domain senses and binds the C-terminal of mis-localized outer membrane proteins (OMPs) or periplasmic protein MucE, leading to catalytic activation of the protease domain. While AlgW is functionally well studied, its exact activation mechanism remains to be elucidated. Here, we show that AlgW is a novel HtrA protease that can be biochemically activated by both peptide and lipid signals. Compared with the corresponding homologue DegS in , AlgW exhibits a distinct substrate specificity and regulation mechanism. Structural, biochemical, and mutagenic analyses revealed that, by specifically binding to the C-terminal decapeptide of MucE, AlgW could adopt more relaxed conformation and obtain higher activity than with tripeptide activation. We also investigated the regulatory mechanism of the L loop in AlgW and proved that the unique structural feature of this region was responsible for the distinct enzymatic property of AlgW. These results demonstrate the unique and diverse activation mechanism of AlgW, which  may utilize to enhance its adaptability to environmental stress. HtrA-family proteases are commonly employed to sense the protein folding stress and activate the regulated intramembrane proteolysis (RIP) cascade in Gram-negative bacteria. Here, we reveal the unique dual-signal activation and dynamic regulation properties of AlgW, an HtrA-type protease triggering the AlgU stress-response pathway, which controls alginate production and mucoid conversion in  The structural and functional data offer insights into the molecular basis underlying the transition of different activation states of AlgW in response to different effectors. Probing these unique features provides an opportunity to correlate the diverse regulation mechanism of AlgW with the high adaptability of  to environmental changes during infection.

Molecular Basis of the Versatile Regulatory Mechanism of HtrA-Type Protease AlgW from Pseudomonas aeruginosa.

Structural insights into sphingosine-1-phosphate recognition and ligand selectivity of S1PR3-Gi signaling complexes.

Lanthipeptides are an important group of natural products with diverse biological functions, and their biosynthesis requires the removal of N-terminal leader peptides (LPs) by designated proteases. LanP enzymes, a subgroup of M1 zinc-metallopeptidases, have been recently identified as bifunctional proteases with both endo- and aminopeptidase activities to remove LPs of class III and class IV lanthipeptides. Herein, we report the biochemical and structural characterization of EryP as the LanP enzyme from the biosynthesis of class III lanthipeptide erythreapeptin. We determined X-ray crystal structures of EryP in three conformational states, the open, intermediate and closed states, and identified a unique interdomain Ca binding site as a regulatory element that modulates its domain dynamics and proteolytic activity. Inspired by this regulatory Ca binding, we developed a strategy to engineer LanP enzymes for enhanced catalytic activities by strengthening interdomain associations and driving the conformational equilibrium toward their closed forms.

Article	Year
Effect of strontium ions on the growth of ROS17/2.8 cells on porous calcium polyphosphate scaffolds. Biomaterials\| PubMed ID: 16143392	2006
Mechanistic insights into the allosteric regulation of aspartate kinase. The Biochemical journal\| PubMed ID: 29382741	2018
Structural and Functional Insights into PpgL, a Metal-Independent β-Propeller Gluconolactonase That Contributes to Virulence. Infection and immunity\| PubMed ID: 30642898	2019
Molecular basis of the lipid-induced MucA-MucB dissociation in Pseudomonas aeruginosa. Communications biology\| PubMed ID: 32747658	2020
Structural and biochemical analysis of 1-Cys peroxiredoxin ScPrx1 from Saccharomyces cerevisiae mitochondria. Biochimica et biophysica acta. General subjects\| PubMed ID: 32805320	2020
Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element. Biochimica et biophysica acta. General subjects\| PubMed ID: 33010351	2021
Structural and biochemical studies of the glycosyltransferase Bs-YjiC from Bacillus subtilis. International journal of biological macromolecules\| PubMed ID: 33152360	2021
Pseudomonas aeruginosa antitoxin HigA functions as a diverse regulatory factor by recognizing specific pseudopalindromic DNA motifs. Environmental microbiology\| PubMed ID: 33346387	2021
Molecular Basis of the Versatile Regulatory Mechanism of HtrA-Type Protease AlgW from Pseudomonas aeruginosa. mBio\| PubMed ID: 33622718	2021
Structural insights into sphingosine-1-phosphate recognition and ligand selectivity of S1PR3-Gi signaling complexes. Cell research\| PubMed ID: 34545189	2022
Conformational remodeling enhances activity of lanthipeptide zinc-metallopeptidases. Nature chemical biology\| PubMed ID: 35513512	2022

Chang Zhao

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