-
Microbial Cell Factories Apr 2021Trans-4-hydroxy-L-proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for... (Review)
Review
Trans-4-hydroxy-L-proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans-4-hydroxy-L-proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low productivity, a complex process and heavy environmental pollution. Presently, trans-4-hydroxy-L-proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to effectively construct microbial cell factories that have improved the trans-4-hydroxy-L-proline biosynthetic pathway. To probe the potential of microorganisms for trans-4-hydroxy-L-proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans-4-hydroxy-L-proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production.
Topics: Bacteria; Hydroxyproline; Metabolic Engineering
PubMed: 33882914
DOI: 10.1186/s12934-021-01579-2 -
Brazilian Journal of Biology = Revista... 2023The objective of this study was to evaluate the physiology and production of sugar-apple as a function of irrigation intervals and foliar application of proline under...
The objective of this study was to evaluate the physiology and production of sugar-apple as a function of irrigation intervals and foliar application of proline under the conditions of Paraíba's semi-arid region. A randomized block design was laid out in a 4 × 2 factorial scheme, with treatments resulting from the combination of four irrigation intervals (1, 4, 8 and 12 days) and two concentrations of proline (0 and 10 mM), with four replicates, and the plot consisted of four usable plants. Increase in irrigation intervals reduced the gas exchange of sugar-apple plants at 298 days after transplanting. Exogenous application of proline at concentration of 10 mM increased contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids and fruit fresh mass in plants grown under 12-day irrigation intervals.
Topics: Chlorophyll A; Dehydration; Malus; Proline; Sugars
PubMed: 37255204
DOI: 10.1590/1519-6984.273404 -
Essays in Biochemistry Sep 2019Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the... (Review)
Review
Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour.
Topics: Animals; Collagen; Humans; Hydroxylation; Hydroxyproline; Integrins; Procollagen-Proline Dioxygenase; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Quaternary
PubMed: 31350381
DOI: 10.1042/EBC20180053 -
Amino Acids Dec 2021In the 35 years since the introduction of the "proline cycle", its relevance to human tumors has been widely established. These connections are based on a variety of...
In the 35 years since the introduction of the "proline cycle", its relevance to human tumors has been widely established. These connections are based on a variety of mechanisms discovered by many laboratories and have stimulated the search for small molecule inhibitors to treat cancer or metastases. In addition, the multi-layered connections of the proline cycle and the role of proline and hydroxyproline in collagen provide an important regulatory link between the extracellular matrix and metabolism.
Topics: Collagen; Extracellular Matrix; Humans; Hydroxyproline; Neoplasms; Proline; Small Molecule Libraries
PubMed: 34825974
DOI: 10.1007/s00726-021-03103-7 -
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 -
Molecular Pharmacology Apr 2022The WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase... (Review)
Review
The WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have well established functions in the maintenance of cell volume and ion homeostasis. Mutations in these kinases have been linked to an inherited form of hypertension, neurologic defects, and other pathologies. A rapidly expanding body of evidence points to the involvement of WNKs in regulating multiple diverse cellular processes as well as the progression of some forms of cancer. How OSR1 and SPAK contribute to these processes is well understood in some cases but completely unknown in others. OSR1 and SPAK are targeted to both WNKs and substrates via their conserved C-terminal (CCT) protein interaction domains. Considerable effort has been put forth to understand the structure, function, and interaction specificity of the CCT domains in relation to WNK signaling, and multiple inhibitors of WNK signaling target these domains. The domains bind RFxV and RxFxV protein sequence motifs with the consensus sequence R-F-x-V/I or R-x-F-x-V/I, but residues outside the core motif also contribute to specificity. CCT interactions are required for OSR1 and SPAK activation and deactivation as well as cation-chloride cotransporter substrate phosphorylation. All four WNKs also contain CCT-like domains that have similar structures and conserved binding residues when compared with CCT domains, but their functions and interaction specificities are mostly unknown. A better understanding of the varied actions of these domains and their interactions will better define the known signaling mechanisms of the WNK pathway as well as uncover new ones. SIGNIFICANCE STATEMENT: WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have been shown to be involved in an array of diverse cellular processes. Here we review the function of modular protein interaction domains found in OSR1 and SPAK as well as related domains found in WNKs.
Topics: Alanine; Proline; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases; Signal Transduction
PubMed: 34312216
DOI: 10.1124/molpharm.121.000307 -
Cells Nov 2023The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo...
The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with tetramethylrhodamine methyl ester and 2',7'-dichlorofluorescein diacetate showed that the culture of embryos in the presence of Pro, or either of these analogues, reduced mitochondrial activity and reactive oxygen species (ROS), respectively, indicating potential mechanisms by which embryo development is improved. Inhibition of the Pro metabolism enzyme, proline oxidase, by tetrahydro-2-furoic-acid prevented these reductions and concomitantly prevented the improved development. The ways in which Pro, PA and L4T reduce mitochondrial activity and ROS appear to differ, despite their structural similarity. Specifically, the results are consistent with Pro reducing ROS by reducing mitochondrial activity while PA and L4T may be acting as ROS scavengers. All three may work to reduce ROS by contributing to the GSH pool. Overall, our results indicate that reduction in mitochondrial activity and oxidative stress are potential mechanisms by which Pro and its analogues act to improve pre-implantation embryo development.
Topics: Animals; Mice; Reactive Oxygen Species; Proline; Oxidative Stress; Blastocyst; Embryonic Development
PubMed: 37998375
DOI: 10.3390/cells12222640 -
Microbial Biotechnology Mar 2021Trans-4-hydroxy-l-proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging...
Trans-4-hydroxy-l-proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging for industrial production of Hyp due to heavy environmental pollution and low production efficiency. To establish a green and efficient process for Hyp production, the proline 4-hydroxylase (DsP4H) from Dactylosporangium sp. RH1 was overexpressed and functionally characterized in Escherichia coli BL21(DE3). The recombinant DsP4H with l-proline as a substrate exhibited K , k and k /K values up to 0.80 mM, 0.52 s and 0.65 s ·mM respectively. Furthermore, DsP4H showed the highest activity at 35°C and pH 6.5 towards l-proline. The highest enzyme activity of 175.6 U mg was achieved by optimizing culture parameters. Under the optimal transformation conditions in a 5-l fermenter, Hyp titre, conversion rate and productivity were up to 99.9 g l , 99.9% and 2.77 g l h respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.
Topics: Bacterial Outer Membrane Proteins; Bioreactors; Escherichia coli; Escherichia coli Proteins; Hydroxyproline; Proline; Prolyl Hydroxylases
PubMed: 32618422
DOI: 10.1111/1751-7915.13616 -
Genetics May 2021The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to...
The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.
Topics: Animals; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mutation; Phenotype; Proline; Pyrroline Carboxylate Reductases
PubMed: 33734376
DOI: 10.1093/genetics/iyab048 -
Cancer Research Communications Nov 2023IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We...
UNLABELLED
IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFNγ-treated colorectal cancer patient-derived organoids and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNγ. IFNγ-induced changes in the abundance of source proteins, switching from the constitutive to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is upregulated or downregulated on HLA-I through IFNγ. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFNγ is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics.
SIGNIFICANCE
IFNγ remodels the HLA-I-presented immunopeptidome. We showed that peptide-specific factors influence whether a peptide is upregulated or downregulated and identified a preferential loss or downregulation of those with proline near the peptide center. This will help selecting immunotherapy target antigens which are consistently presented by cancer cells.
Topics: Humans; Proteomics; Neoplasms; Interferon-gamma; Antigens; Peptides; Proline
PubMed: 37991387
DOI: 10.1158/2767-9764.CRC-23-0121