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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 -
The Journal of Organic Chemistry May 2019Organocatalysis is an emerging field, in which small metal-free organic structures catalyze a diversity of reactions with a remarkable stereoselectivity. The ability to...
Organocatalysis is an emerging field, in which small metal-free organic structures catalyze a diversity of reactions with a remarkable stereoselectivity. The ability to selectively switch on such pathways upon demand has proven to be a valuable tool in biological systems. Light as a trigger provides the ultimate spatial and temporal control of activation. However, there have been limited examples of phototriggered catalytic systems. Herein, we describe the synthesis and application of a caged proline system that can initiate organocatalysis upon irradiation. The caged proline was generated using the highly efficient 4-carboxy-5,7-dinitroindolinyl (CDNI) photocleavable protecting group in a four-step synthesis. Advantages of this system include water solubility, biocompatibility, high quantum yield for catalyst release, and responsiveness to two-photon excitation. We showed the light-triggered catalysis of a crossed aldol reaction, a Mannich reaction, and a self-aldol condensation reaction. We also demonstrated light-initiated catalysis, leading to the formation of a biocide in situ, which resulted in the growth inhibition of E. coli, with as little as 3 min of irradiation. This technique can be broadly applied to other systems, by which the formation of active forms of drugs can be catalytically assembled remotely via two-photon irradiation.
Topics: Anti-Bacterial Agents; Catalysis; Escherichia coli; Indoles; Kinetics; Photochemical Processes; Proline; Solubility; Water
PubMed: 30908906
DOI: 10.1021/acs.joc.9b00220 -
Frontiers in Bioscience (Landmark... Jun 2023Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an...
BACKGROUND
Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline isomerization. The knowledge and value of a given proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data.
METHODS
After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types ( ± 4 regions) on forming a -Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the Pro content on model peptides and desired point mutations.
RESULTS
Analysis of NMR spectra prove the dependence of the Pro content on the type of the neighboring amino acid-with special attention on aromatic and positively charged sidechains.
CONCLUSIONS
Our results may benefit the design of protein regions with a given -Pro content, and contribute to a better understanding of the roles and functions of IDPs.
Topics: Isomerism; Intrinsically Disordered Proteins; Proline; Peptides; Magnetic Resonance Spectroscopy; Protein Conformation
PubMed: 37395034
DOI: 10.31083/j.fbl2806127 -
Archives of Biochemistry and Biophysics Feb 2021Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using...
Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone.
Topics: Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Glutamate-5-Semialdehyde Dehydrogenase; Hydroxyproline; Membrane Proteins; Proline; Protein Binding; Sinorhizobium meliloti; Stereoisomerism
PubMed: 33333077
DOI: 10.1016/j.abb.2020.108727 -
Biomolecules Oct 2021Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and... (Review)
Review
Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.
Topics: Diketopiperazines; Dipeptides; Drug Discovery; Humans; Neoplasms; Peptides, Cyclic; Proline
PubMed: 34680148
DOI: 10.3390/biom11101515 -
Amino Acids Jan 2010The central effects of L-proline, D-proline and trans-4-hydroxy-L-proline were investigated by using the acute stressful model with neonatal chicks in Experiment 1....
The central effects of L-proline, D-proline and trans-4-hydroxy-L-proline were investigated by using the acute stressful model with neonatal chicks in Experiment 1. Sedative and hypnotic effects were induced by all compounds, while plasma corticosterone release under isolation stress was only attenuated by L-proline. To clarify the mechanism by which L-proline and D-proline induce sedative and hypnotic effects, the contribution of the strychnine-sensitive glycine receptor (glycine receptor) and N-methyl-D-aspartate glutamate receptor (NMDA receptor) were further investigated. In Experiments 2-3, the glycine receptor antagonist strychnine was co-injected intracerebroventricular (i.c.v.) with L-proline or D-proline. The suppression of isolation-induced stress behavior by D-proline was attenuated by strychnine. However, the suppression of stress behavior by L-proline was not attenuated. In Experiment 4, the NMDA receptor antagonist (+)-MK-801 was co-injected i.c.v. with L-proline. The suppression of stress behavior by L-proline was attenuated by (+)-MK-801. These results indicate that L-proline and D-proline differentially induce sedative and hypnotic effects through NMDA and glycine receptors, respectively.
Topics: Animals; Behavior, Animal; Chickens; Hypnotics and Sedatives; Injections; Injections, Intraventricular; Male; Proline; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Stress, Physiological; Vocalization, Animal
PubMed: 19023642
DOI: 10.1007/s00726-008-0204-9 -
Experimental Biology and Medicine... Mar 2016The amino acids in the placenta have multiple functions; however, the therapeutic effects of proline remain poorly for relief postmenopausal symptoms. The aim of present...
The amino acids in the placenta have multiple functions; however, the therapeutic effects of proline remain poorly for relief postmenopausal symptoms. The aim of present study was to evaluate the effects of proline in the treatment of menopause using in vitro and in vivo models. We assessed the therapeutic effects and regulatory mechanisms of proline by using MCF-7 estrogen-dependent cells, MG63 osteoblast cells, and ovariectomized mice model. An in vivo study was carried out in eight-week-old sham and ovariectomized group. The ovariectomized mouse was further subdivided into two groups administered orally with 17β-estradiol or proline (10 mg/kg/day) for eight weeks. Proline significantly increased cell proliferation and Ki-67 levels in MCF-7 cells and enhanced cell proliferation, alkaline phosphatase activity, extracellular signal-regulated kinase phosphorylation, and glutamyl-prolyl-tRNA synthetase activation in MG63 cells. The estrogen receptor-β and estrogen-response elements luciferase activity were significantly increased by proline in MCF-7 and MG63 cells. In ovariectomized mice, oral administration of proline (10 mg/kg/day) for eight weeks significantly reduced body and vaginal weights. Proline also significantly increased serum estradiol and alkaline phosphatase levels, whereas serum luteinizing hormone was decreased by proline. In addition, detailed microcomputed tomography analysis showed that the proline notably enhanced bone mineral density, trabecular bone volume, and trabecular number in ovariectomized mice. Those findings implied that proline can be a promising candidate for the treatment of menopause.
Topics: Administration, Oral; Animals; Disease Models, Animal; Female; Humans; Menopause; Mice, Inbred BALB C; Models, Biological; Osteoporosis, Postmenopausal; Proline; Treatment Outcome
PubMed: 26830682
DOI: 10.1177/1535370216629011 -
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 -
Organic & Biomolecular Chemistry Jan 2022Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ-pyrroline-5-carboxylate. PRODH plays a...
Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ-pyrroline-5-carboxylate. PRODH plays a central role in the metabolic rewiring of cancer cells, which has motivated the discovery of inhibitors. Here, we studied the inhibition of PRODH by 18 proline-like compounds to understand the structural and chemical features responsible for the affinity of the best-known inhibitor, -(-)-tetrahydro-2-furoic acid (1). The compounds were screened, and then six were selected for more thorough kinetic analysis: cyclobutane-1,1-dicarboxylic acid (2), cyclobutanecarboxylic acid (3), cyclopropanecarboxylic acid (4), cyclopentanecarboxylic acid (16), 2-oxobutyric acid (17), and (2)-oxetane-2-carboxylic acid (18). These compounds are competitive inhibitors with inhibition constants in the range of 1.4-6 mM, compared to 0.3 mM for 1. Crystal structures of PRODH complexed with 2, 3, 4, and 18 were determined. All four inhibitors bind in the proline substrate site, but the orientations of their rings differ from that of 1. The binding of 3 and 18 is accompanied by compression of the active site to enable nonpolar contacts with Leu513. Compound 2 is unique in that the additional carboxylate displaces a structurally conserved water molecule from the active site. Compound 18 also destabilizes the conserved water, but by an unexpected non-steric mechanism. The results are interpreted using a chemical double mutant thermodynamic cycle. This analysis revealed unanticipated synergism between ring size and hydrogen bonding to the conserved water. These structure-affinity relationships provide new information relevant to the development of new inhibitor design strategies targeting PRODH.
Topics: Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Molecular Structure; Proline; Proline Oxidase; Structure-Activity Relationship
PubMed: 35018940
DOI: 10.1039/d1ob02328d -
Protein Engineering, Design & Selection... Feb 2022Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its...
Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72 Å resolution. Minimal PRODHs should be useful in chemical probe discovery.
Topics: Humans; Proline Oxidase; Proline; Bacterial Proteins
PubMed: 36448708
DOI: 10.1093/protein/gzac016