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The FEBS Journal Jul 2021Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the...
Linker for activation in T cells (LAT) is a critical regulator of T-cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane, is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and super-resolution imaging and flow cytometry, we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics are further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of nonpalmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.
Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Calcium; Cell Membrane; Glycine; Humans; Jurkat Cells; Membrane Proteins; Microscopy, Confocal; Microscopy, Interference; Molecular Dynamics Simulation; Mutation; Proline; Protein Domains; Protein Structure, Secondary; Sequence Homology, Amino Acid; T-Lymphocytes
PubMed: 33458942
DOI: 10.1111/febs.15713 -
Microbial Cell Factories Aug 2022In recent years, there has been a growing demand for microbial production of trans-4-hydroxy-L-proline (t4Hyp), which is a value-added amino acid and has been widely...
BACKGROUND
In recent years, there has been a growing demand for microbial production of trans-4-hydroxy-L-proline (t4Hyp), which is a value-added amino acid and has been widely used in the fields of medicine, food, and cosmetics. In this study, a multivariate modular metabolic engineering approach was used to remove the bottleneck in the synthesis pathway of t4Hyp.
RESULTS
Escherichia coli t4Hyp synthesis was performed using two modules: a α-ketoglutarate (α-KG) synthesis module (K module) and L-proline synthesis with hydroxylation module (H module). First, α-KG attrition was reduced, and then, L-proline consumption was inhibited. Subsequently, to improve the contribution to proline synthesis with hydroxylation, optimization of gene overexpression, promotor, copy number, and the fusion system was performed. Finally, optimization of the H and K modules was performed in combination to balance metabolic flow. Using the final module H1K4 in a shaking flask culture, 8.80 g/L t4Hyp was produced, which was threefold higher than that produced by the W0 strain.
CONCLUSIONS
These strategies demonstrate that a microbial cell factory can be systematically optimized by modular engineering for efficient production of t4Hyp.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Hydroxyproline; Ketoglutaric Acids; Metabolic Engineering; Proline
PubMed: 35953819
DOI: 10.1186/s12934-022-01884-4 -
Biochemistry Jun 2018Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival,...
Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ-pyrroline-5-carboxylate (P5C), which forms l-glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD-dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline-P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline-P5C cycle has been implicated in supporting ATP production, protein and nucleotide synthesis, anaplerosis, and redox homeostasis in cancer cells. This Perspective details the structures and reaction mechanisms of PRODH and PYCR and the role of the proline-P5C cycle in cancer metabolism. A major challenge in the field is to discover inhibitors that specifically target PRODH and PYCR isoforms for use as tools for studying proline metabolism and the functions of the proline-P5C cycle in cancer. These molecular probes could also serve as lead compounds in cancer drug discovery targeting the proline-P5C cycle.
Topics: Animals; Biosynthetic Pathways; Cell Proliferation; Humans; Molecular Docking Simulation; Neoplasms; Oxidation-Reduction; Proline; Proline Oxidase; Pyrroline Carboxylate Reductases; delta-1-Pyrroline-5-Carboxylate Reductase
PubMed: 29648801
DOI: 10.1021/acs.biochem.8b00215 -
Journal of Hepatology Nov 2014The establishment of robust HCV cell culture systems and characterization of the viral life cycle provided the molecular basis for highly innovative, successful years in... (Review)
Review
The establishment of robust HCV cell culture systems and characterization of the viral life cycle provided the molecular basis for highly innovative, successful years in HCV drug development. With the identification of direct-acting antiviral agents (DAAs), such as NS3/4A protease inhibitors, NS5A replication complex inhibitors, nucleotide and non-nucleoside polymerase inhibitors, as well as host cell targeting agents, novel therapeutic strategies were established and competitively entered clinical testing. The first-in-class NS3/4A protease inhibitors telaprevir and boceprevir, approved in 2011, were recently outpaced by the pan-genotypic nucleotide polymerase inhibitor sofosbuvir that in combination with pegylated interferon and ribavirin, further shortens therapy durations and also offers the first interferon-free HCV treatment option. In the challenging race towards the goal of interferon-free HCV therapies, however, several oral DAA regimens without nucleotide polymerase inhibitors that combine a NS3/4A protease inhibitor, a NS5A inhibitor and/or a non-nucleoside polymerase inhibitor yielded competitive results. Second generation NS3/4A protease and NS5A inhibitors promise an improved genotypic coverage and a high resistance barrier. Results of novel DAA combination therapies without the backbone of a nucleotide polymerase inhibitor, as well as treatment strategies involving host targeting agents are reviewed herein.
Topics: Antiviral Agents; Drug Therapy, Combination; Genotype; Hepacivirus; Hepatitis C; Humans; Interferons; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Proline; Protease Inhibitors; Viral Nonstructural Proteins
PubMed: 25443350
DOI: 10.1016/j.jhep.2014.08.014 -
Journal of the American Society For... Aug 2023The fragmentation characteristics of ions produced from proline-containing heptapeptides have been studied in detail. The study has utilized the following C-terminally...
The fragmentation characteristics of ions produced from proline-containing heptapeptides have been studied in detail. The study has utilized the following C-terminally amidated model peptides: PA, APA, APA, APA, APA, APA, AP, PYAGFLV, PAGFLVY, PGFLVYA, PFLVYAG, PLVYAGF, PVYAGFL, YPAGFLV, YAPGFLV, YAGPFLV, YAGFPLV, YAGFLPV, YAGFLVP, PYAFLVG, PVLFYAG, APXA, and AXPA (where X = C, D, F, G, L, V, and Y, respectively). The results have shown that ions undergo head-to-tail cyclization and form a macrocyclic structure. Under the collision-induced dissociation (CID) condition, it generates nondirect sequence ions regardless of the position of the proline and the neighboring amino acid residues. This study highlights the unusual and unique fragmentation behavior of proline-containing heptapeptides. Following the head-to-tail cyclization, the ring opens up and places the proline residue in the N-terminal position while forming a regular oxazolone form of ions for all peptide series. Then, the fragmentation reaction pathway is followed by the elimination of proline with its C-terminal neighbor residue as an oxazolone (e.g., PX) for all proline-containing peptide series.
Topics: Oxazolone; Proline; Peptides; Ions; Cyclization
PubMed: 37402129
DOI: 10.1021/jasms.3c00049 -
Journal of Bacteriology Feb 2016Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and...
UNLABELLED
Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ(1)-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ(1)-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes.
IMPORTANCE
Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Hydroxyproline; Models, Molecular; Molecular Structure; Oxidoreductases Acting on CH-NH Group Donors; Proline; Protein Conformation; Recombinant Proteins; Sinorhizobium meliloti
PubMed: 26833407
DOI: 10.1128/JB.00961-15 -
Lancet (London, England) Feb 2022
Topics: COVID-19 Testing; Developing Countries; Drug Combinations; Global Health; Health Services Accessibility; Humans; Lactams; Leucine; Nitriles; Proline; Ritonavir; COVID-19 Drug Treatment
PubMed: 35219388
DOI: 10.1016/S0140-6736(22)00372-5 -
International Journal of Molecular... Dec 2022Carbon nanoparticles have potential threats to plant growth and stress tolerance. The polyhydroxy fullerene-fullerol (one of the carbon nanoparticles) could increase...
Carbon nanoparticles have potential threats to plant growth and stress tolerance. The polyhydroxy fullerene-fullerol (one of the carbon nanoparticles) could increase biomass accumulation in several plants subjected to drought; however, the underlying molecular and metabolic mechanisms governed by fullerol in improving drought tolerance in remain unclear. In the present study, exogenous fullerol was applied to the leaves of seedlings under drought conditions. The results of transcriptomic and metabolomic analyses revealed changes in the molecular and metabolic profiles of . The differentially expressed genes and the differentially accumulated metabolites, induced by drought or fullerol treatment, were mainly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbohydrate metabolism (e.g., "carbon metabolism" and "galactose metabolism"), amino acid metabolism (e.g., "biosynthesis of amino acids" and "arginine and proline metabolism"), and secondary metabolite metabolism (e.g., "biosynthesis of secondary metabolites"). For carbohydrate metabolism, the accumulation of oligosaccharides (e.g., sucrose) was decreased, whereas that of monosaccharides (e.g., mannose and myo-inositol) was increased by drought. With regard to amino acid metabolism, under drought stress, the accumulation of amino acids such as phenylalanine and tryptophan decreased, whereas that of glutamate and proline increased. Further, for secondary metabolite metabolism, subjected to soil drying showed a reduction in phenolics and flavonoids, such as hyperoside and trans-3-coumaric acid. However, the accumulation of carbohydrates was almost unchanged in fullerol-treated subjected to drought. When exposed to water shortage, the accumulation of amino acids, such as proline, was decreased upon fullerol treatment. However, that of phenolics and flavonoids, such as luteolin and trans-3-coumaric acid, was enhanced. Our findings suggest that fullerol can alleviate the inhibitory effects of drought on phenolics and flavonoids to enhance drought tolerance in .
Topics: Brassica napus; Drought Resistance; Stress, Physiological; Droughts; Proline; Carbon; Gene Expression Regulation, Plant
PubMed: 36499633
DOI: 10.3390/ijms232315304 -
Journal of Bacteriology Jun 2022The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite...
The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of , it inhibited the growth of the deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.
Topics: Animals; Arginine; Carbon; Glutamic Acid; Mutation; Proline; Ribose; Staphylococcal Infections; Staphylococcus aureus
PubMed: 35546540
DOI: 10.1128/jb.00018-22 -
Applied and Environmental Microbiology Apr 2018Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: -4-hydroxy-l-proline and, in most echinocandin...
Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: -4-hydroxy-l-proline and, in most echinocandin structures, (-2,3)-3-hydroxy-(-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by , both amino acids are found in pneumocandin A, while in pneumocandin B the latter residue is replaced by -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A and B are produced by the fungus They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A and B, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in produces the same hydroxyprolines, with an increased proportion of -3-hydroxyproline. However, echinocandin B biosynthesis does not require -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in and provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.
Topics: Antifungal Agents; Ascomycota; Aspergillus; Echinocandins; Fungal Proteins; Hydroxyproline
PubMed: 29352089
DOI: 10.1128/AEM.02370-17