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Nature Communications Nov 2020Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by...
Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for α-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of α-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins.
Topics: Amyloid; Catalysis; Cyclophilin A; Cyclosporine; Humans; Isomerism; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Chaperones; Parkinson Disease; Proline; Protein Aggregates; Protein Binding; Protein Domains; alpha-Synuclein
PubMed: 33247146
DOI: 10.1038/s41467-020-19844-0 -
Cell Metabolism Nov 2016The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy...
The role of essential amino acids in metabolic reprogramming of cancer cells is now well established, whereas the role of non-essential amino acids (NEAAs) in malignancy remains less clear. Here, we have identified an important role for the NEAA proline in the tumorigenic potential of a subset of cancer cells. By profiling a large panel of cancer cell lines, we observed that proline consumption and expression of proline biosynthesis enzymes were well correlated with clonogenic and tumorigenic potential. Moreover, proline starvation or inhibition of proline biosynthesis enzymes impaired clonogenic/tumorigenic potential. Cancer cells exhibiting dependency on exogenous proline displayed hyperactivation of the mTORC1-mediated 4EBP1 signaling axis, as well as unresolved ER stress. Exogenous proline alleviated ER stress and promoted cellular homeostasis and clonogenicity. Increased dependence on proline may therefore define a specific vulnerability in some cancers that can be exploited by proline depletion.
Topics: Animals; Carcinogenesis; Cell Line; Cell Proliferation; Clone Cells; Endoplasmic Reticulum Stress; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphoproteins; Proline; Protein Biosynthesis; RNA Caps; TOR Serine-Threonine Kinases
PubMed: 27618686
DOI: 10.1016/j.cmet.2016.08.008 -
Journal of Experimental Botany Feb 2024This article comments on: Zheng Y, Cabassa-Hourton C, Eubel H, Chevreux G, Lignieres L, Crilat E, Braun H-P, Lebreton S, Savouré A. 2024. Pyrroline-5-carboxylate...
This article comments on: Zheng Y, Cabassa-Hourton C, Eubel H, Chevreux G, Lignieres L, Crilat E, Braun H-P, Lebreton S, Savouré A. 2024. Pyrroline-5-carboxylate metabolism protein complex detected in leaf mitochondria. Journal of Experimental Botany 75, 917–934.
Topics: Mitochondria; Proline
PubMed: 38307518
DOI: 10.1093/jxb/erad446 -
Molecular Biology and Evolution Mar 2023Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which,...
Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.
Topics: Cell Survival; Biosynthetic Pathways; Mutation; Biological Evolution; Proline
PubMed: 36788592
DOI: 10.1093/molbev/msad032 -
American Journal of Respiratory Cell... Nov 2019Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil...
Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil chemoattractant proline-glycine-proline (PGP) and its more potent acetylated form (acPGP) have been found to be elevated in patients with COPD and act via CXCR2. Here, we investigated the impact of neutralizing PGP peptides in a murine model for emphysema. The PGP-neutralizing peptide l-arginine-threonine-arginine (RTR) was used first in a 6-week model of cigarette smoke exposure, where it attenuated lung inflammation. Then, in a model of chronic smoke exposure, mice were exposed to cigarette smoke and RTR treatment was initiated after 10 weeks of smoke exposure. This treatment was continued together with smoke exposure for another 13 weeks, for a total of 23 weeks of smoke exposure. RTR significantly inhibited neutrophil and macrophage influx into the lungs in the 6-week model of exposure. RTR also attenuated the development of emphysema, normalized lung volumes, and reduced right ventricular hypertrophy in the chronic exposure model. Murine epithelia expressed CXCR2, and this expression was increased after smoke exposure. , human bronchial epithelial cells also demonstrated robust expression of CXCR2, and stimulation of primary human bronchial epithelial cells with acPGP led to increased release of MMP-9 and IL-8. Overall, these results provide evidence that acPGP plays a critical role during the development of emphysema in cigarette smoke-induced injury, and highlight a new epithelial mechanism by which acPGP augments neutrophilic inflammation.
Topics: Animals; Cells, Cultured; Humans; Inflammation; Lung; Mice; Neutrophils; Oligopeptides; Proline; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Smoke
PubMed: 30958968
DOI: 10.1165/rcmb.2018-0216OC -
Proceedings of the National Academy of... May 2021Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic...
Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid-liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.
Topics: Binding Sites; Blood Proteins; Carbohydrates; Galectin 3; Galectins; Glycosylation; Humans; Polysaccharides; Proline; Protein Binding
PubMed: 33952698
DOI: 10.1073/pnas.2021074118 -
The Journal of Biological Chemistry Mar 2022Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In...
Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.
Topics: Amino Acid Oxidoreductases; Animals; Chick Embryo; Escherichia coli; HEK293 Cells; Humans; Hydroxybutyrate Dehydrogenase; Hydroxyproline; Mammals; Proline; Rabbits; Rats
PubMed: 35150746
DOI: 10.1016/j.jbc.2022.101708 -
Microbial Biotechnology Mar 2021Microbial production of trans-4-hydroxy-l-proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for...
Microbial production of trans-4-hydroxy-l-proline (Hyp) offers significant advantages over conventional chemical extraction. However, it is still challenging for industrial production of Hyp due to its low production efficiency. Here, chassis engineering was used for tailoring Escherichia coli cellular metabolism to enhance enzymatic production of Hyp. Specifically, four proline 4-hydroxylases (P4H) were selected to convert l-proline to Hyp, and the recombinant strain overexpressing DsP4H produced 32.5 g l Hyp with α-ketoglutarate addition. To produce Hyp without α-ketoglutarate addition, α-ketoglutarate supply was enhanced by rewiring the TCA cycle and l-proline degradation pathway, and oxygen transfer was improved by fine-tuning heterologous haemoglobin expression. In a 5-l fermenter, the engineered strain E. coliΔsucCDΔputA-VHb -DsP4H showed a significant increase in Hyp titre, conversion rate and productivity up to 49.8 g l , 87.4% and 1.38 g l h respectively. This strategy described here provides an efficient method for production of Hyp, and it has a great potential in industrial application.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Hydroxyproline; Metabolic Engineering; Proline; Prolyl Hydroxylases
PubMed: 32396278
DOI: 10.1111/1751-7915.13573 -
The Journal of Organic Chemistry Nov 2022The asymmetric synthesis of bicyclic highly substituted tetrahydropyrans is described. The reaction is catalyzed by unnatural γ-dipeptides based on densely substituted...
The asymmetric synthesis of bicyclic highly substituted tetrahydropyrans is described. The reaction is catalyzed by unnatural γ-dipeptides based on densely substituted l- and d-proline derivatives. This organocatalytic one-pot reaction takes place among a ketone, a nitroalkene, and an aldehyde to yield an octahydro-2-chromene scaffold. Monomeric species, from which the corresponding γ-dipeptides are synthesized, cannot catalyze the reaction, thus confirming the emergent nature of the catalytic behavior of these dimeric species.
Topics: Dipeptides; Proline; Catalysis; Ketones; Aldehydes
PubMed: 36178434
DOI: 10.1021/acs.joc.2c01230 -
Microbiology (Reading, England) Oct 2014L-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the...
L-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived L-proline derivatives N-methyl-L-proline, L-proline betaine (stachydrine), trans-4-L-hydroxproline and trans-4-hydroxy-L-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-L-hydroxyproline and N-methyl-L-proline was unable to do so. Except for L-proline, none of these compounds served as a nutrient for B. subtilis. L-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the L-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that L-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. L-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: L-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC-betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with L-proline betaine.
Topics: Bacillus subtilis; Hot Temperature; Osmotic Pressure; Phytochemicals; Plants; Proline; Stress, Physiological
PubMed: 25012968
DOI: 10.1099/mic.0.079665-0