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Bruck syndrome is an autosomal recessive form of osteogenesis imperfecta caused by biallelic variants in PLOD2 or FKBP10 and is characterized by joint contractures, bone fragility, short stature, and scoliosis. PLOD2 encodes LH2, which hydroxylates type I collagen telopeptide lysines, a critical step for collagen crosslinking. The Plod2 global knockout mouse model is limited by early embryonic lethality, and thus, the role of PLOD2 in skeletogenesis is not well understood. We generated a novel Plod2 mouse line modeling a variant identified in two unrelated individuals with Bruck syndrome: PLOD2 c.1559dupC, predicting a frameshift and loss of the long isoform LH2b. In the mouse, the duplication led to loss of LH2b mRNA as well as significantly reduced total LH2 protein. This model, Plod2fs/fs, survived up to E18.5 although in non-Mendelian genotype frequencies. The homozygous frameshift model recapitulated the joint contractures seen in Bruck syndrome and had indications of absent type I collagen telopeptide lysine hydroxylation in bone. Genetically labeling tendons with Scleraxis-GFP in Plod2fs/fs mice revealed the loss of extensor tendons in the forelimb by E18.5, and developmental studies showed extensor tendons developed through E14.5 but were absent starting at E16.5. Second harmonic generation showed abnormal tendon type I collagen fiber organization, suggesting structurally abnormal tendons. Characterization of the skeleton by μCT and Raman spectroscopy showed normal bone mineralization levels. This work highlights the importance of properly crosslinked type I collagen in tendon and bone, providing a promising new mouse model to further our understanding of Bruck syndrome. Lay Summary: Bruck syndrome is a rare disease where individuals have brittle bone as well as contracted or stiff joints. Mutations in two genes are associated with Bruck syndrome and, in this work, we focus on PLOD2. Mice without Plod2 die at an early embryonic stage, before they have a chance to fully develop. In this work, we created a mouse with a PLOD2 mutation seen in people with Bruck syndrome. Some of these new Bruck syndrome model mice survived to a later gestational age, but all died at birth. The Bruck syndrome mice were small and had contracted joints. We found that they were missing tendons in their arms and had structurally abnormal tendons in their knees. Bone mineralization was normal, but there were indications that the modifications needed for normal type I collagen structure were absent. Overall, this is an advantageous new mouse model of Bruck syndrome that can be used to study this rare disease and highlights the importance of Plod2 in tendon. [ABSTRACT FROM AUTHOR]

Amino alcohols are important compounds that are widely used in the polymer and pharmaceutical industry, particularly when used as chiral scaffolds in organic synthesis. The hydroxylation of polyamide polymers may allow crosslinking between molecular chains through the esterification reactions of hydroxyl and carboxyl groups. Therefore, this may alter the functional properties of polyamide polymers. 2-hydroxycadaverine (2HyC), as a new type of chiral amino alcohol, has potential applications in the pharmaceutical, chemical, and polymer industries. Currently, 2HyC production has only been realized via pure enzyme catalysis or two-stage whole-cell biocatalysis, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2HyC in industrial applications. Here, we designed and constructed a promising artificial pathway in Escherichia coli for producing 2HyC from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs two enzymes to sequentially convert lysine into 2HyC. However, the catalytic activity of wild-type pyridoxal phosphate-dependent decarboxylase from Chitinophage pinensis (DCCp) toward 3-hydroxylysine is lower, resulting in the lower production of 2HyC. Thus, the higher catalytic activity of DCCp is desired for low-cost and expanded industrial applications of 2HyC. To improve the catalytic activity of DCCp, a mutant library of DCCp was first built using a semi-rational design. The Kcat/Km of mutant DCCp (R53D/V94I) increased by 63%. A titer of 359 mg/L 2HyC was produced in shake flasks, with a 2HyC titer increase of 54% compared to control strain ML101. The results show that the production of 2HyC was effectively increased through a semi-rational design strategy. These findings lay the foundation for the development and utilization of renewable resources to produce 2HyC in microorganisms via an efficient, green, and sustainable biosynthetic strategy for further industrial application. [ABSTRACT FROM AUTHOR]

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases. [ABSTRACT FROM AUTHOR]

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

Histamine from our diet or gut microbes can trigger gastrointestinal disturbances, and resistant potato starch (RPS) has previously been shown to alleviate these symptoms while increasing levels of health-associated bacteria such as Akkermansia through unknown mechanisms. Post hoc exploratory metabolomic analysis of serum amino acid, amine, and carnitine metabolites in participants consuming 3.5 g/day RPS or placebo (n = 48) was performed using liquid chromatography-mass spectrometry to determine whether RPS positively influences histamine metabolism and related parameters. Histamine levels were significantly reduced by RPS treatment, but histamine-degrading enzyme products were unaffected by RPS. RPS also reduced histamine-secreting Haemophilus and Lactobacillus. Further, metabolites associated with intestinal permeability, including 5-hydroxylysine, acetylspermidine, and short- and medium-chain carnitines ratios, were significantly reduced by RPS treatment, suggesting decreased serum histamine might be related to enhanced gut barrier function. These metabolomic findings expand the value of supplementing the diet with RPS.

Background: Anterior cruciate ligament (ACL) injury is a common disease in clinical practice that seriously affects the daily life of patients. Purpose: To explore the molecular imaging basis of "diminution sign on dual-energy colour mapping" for the diagnosis of ACL injury by dual-energy computed tomography (DECT). Material and Methods: The hydroxylysine and hydroxyproline reagents were prepared in different concentrations. The grouping was shown as follows: a simple concentration change group of an amino acid (group 1/2); a mixed solution group with the concentration increasing synchronously (group 3); a mixed solution group with the concentration reverse increasing and decreasing (group 4); and a mixed solution group that fix one amino acid with increasing concentration of the other (group 5/6). The samples were scanned by DECT. The solution CT value and image signal-to-noise ratio were analyzed. Results: In group 1/2, the brightness of the dual-energy color mapping of each test tube solution and the CT value increased with increasing the concentration of amino acid. In group 6, there was no significant change in the brightness and brilliance of the dual-energy color mapping and the CT value. The remaining three groups showed an increase in the brightness and brilliance of the dual-energy color mapping and the CT value, and this increase was positively associated with the hydroxylysine concentration. Conclusion: The dual-energy staining of the DECT imaging in "tendon" mode is related to hydroxylysine and hydroxyproline. Moreover, the degree of dual-energy color mapping is positively correlated with the change of CT value. [ABSTRACT FROM AUTHOR]

Hydroxy amino acids (HAAs) are of unique value in the chemical and pharmaceutical industry with antiviral, antifungal, antibacterial, and anticancer properties. At present, the hydroxylated amino acids most studied are tryptophan, lysine, aspartic acid, leucine, proline, etc., and some of their derivatives. The hydroxylation of amino acids is inextricably linked to the catalysis of various biological enzymes, such as tryptophan hydroxylase, L-pipecolic acid trans-4-hydroxylase, lysine hydroxylase, etc. Hydroxylase conspicuously increases the variety of amino acid derivatives. For the manufacture of HAAs, the high regioselectivity biocatalytic synthesis approach is favored over chemical synthesis. Nowadays, the widely used method is to transcribe the hydroxylation pathway of various amino acids, including various catalytic enzymes, into Corynebacterium glutamicum or Escherichia coli for heterologous expression and then produce hydroxyamino acids. In this paper, we systematically reviewed the biosynthetic hydroxylation of aliphatic, heterocyclic, and aromatic amino acids and introduced the basic research and application of HAAs. [ABSTRACT FROM AUTHOR]

Amino alcohols are important compounds that are widely used in the polymer and pharmaceutical industry, particularly when used as chiral scaffolds in organic synthesis. The hydroxylation of polyamide polymers may allow crosslinking between molecular chains through the esterification reactions of hydroxyl and carboxyl groups. Therefore, this may alter the functional properties of polyamide polymers. 2-hydroxycadaverine (2HyC), as a new type of chiral amino alcohol, has potential applications in the pharmaceutical, chemical, and polymer industries. Currently, 2HyC production has only been realized via pure enzyme catalysis or two-stage whole-cell biocatalysis, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2HyC in industrial applications. Here, we designed and constructed a promising artificial pathway in Escherichia coli for producing 2HyC from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs two enzymes to sequentially convert lysine into 2HyC. However, the catalytic activity of wild-type pyridoxal phosphate-dependent decarboxylase from Chitinophage pinensis (DCCp) toward 3-hydroxylysine is lower, resulting in the lower production of 2HyC. Thus, the higher catalytic activity of DCCp is desired for low-cost and expanded industrial applications of 2HyC. To improve the catalytic activity of DCCp, a mutant library of DCCp was first built using a semi-rational design. The Kcat/Km of mutant DCCp (R53D/V94I) increased by 63%. A titer of 359 mg/L 2HyC was produced in shake flasks, with a 2HyC titer increase of 54% compared to control strain ML101. The results show that the production of 2HyC was effectively increased through a semi-rational design strategy. These findings lay the foundation for the development and utilization of renewable resources to produce 2HyC in microorganisms via an efficient, green, and sustainable biosynthetic strategy for further industrial application.

The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation and cancer, on integrin-dependent neutrophil adhesion to fibronectin and the concomitant secretion. Ivermectin did not affect the attachment of neutrophils to the substrate and the reactive oxygen species production but sharply inhibited the adhesion-induced release of hydroxylysine and stimulated the release of phenylalanine and cathepsin G. Hydroxylysine is a product of lysyl hydroxylase, which is overexpressed in tumor cells with an increased ability to invade and metastasize. The inhibition of hydroxylysine release by ivermectin, by analogy, may indicate the suppression of neutrophil invasion into tissue. The increase in the release of phenylalanine in our experiments coincided with the secretion of cathepsin G, which indicates the possible role of this enzyme in the cleavage of phenylalanine. What is the substrate in such a reaction is unknown. We demonstrated that exogenously added angiotensin II (1–8) can serve as a substrate for phenylalanine cleavage. Mass spectrometry revealed the formation of angiotensin II (1–7) in the secretion of neutrophils, which attached to fibronectin in the presence of ivermectin and exogenous angiotensin II (1–8), indicating a possible involvement of ivermectin in the inactivation of angiotensin II. [ABSTRACT FROM AUTHOR]

Hydroxy amino acids (HAAs) are of unique value in the chemical and pharmaceutical industry with antiviral, antifungal, antibacterial, and anticancer properties. At present, the hydroxylated amino acids most studied are tryptophan, lysine, aspartic acid, leucine, proline, etc., and some of their derivatives. The hydroxylation of amino acids is inextricably linked to the catalysis of various biological enzymes, such as tryptophan hydroxylase, L-pipecolic acid trans-4-hydroxylase, lysine hydroxylase, etc. Hydroxylase conspicuously increases the variety of amino acid derivatives. For the manufacture of HAAs, the high regioselectivity biocatalytic synthesis approach is favored over chemical synthesis. Nowadays, the widely used method is to transcribe the hydroxylation pathway of various amino acids, including various catalytic enzymes, into Corynebacterium glutamicum or Escherichia coli for heterologous expression and then produce hydroxyamino acids. In this paper, we systematically reviewed the biosynthetic hydroxylation of aliphatic, heterocyclic, and aromatic amino acids and introduced the basic research and application of HAAs.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column. [ABSTRACT FROM AUTHOR]

Recent studies demonstrate the involvement of inflammatory processes in the development of depression and the anti-inflammatory effects of antidepressants. Infiltration and adhesion of neutrophils to nerve tissues and their aggressive secretion are considered as possible causes of inflammatory processes in depression. We studied the effect of the antidepressant imipramine on the adhesion and accompanied secretion of neutrophils under control conditions and in the presence of lipopolysaccharides (LPS). As a model of integrin-dependent neutrophil infiltration into tissues, we used integrin-dependent adhesion of neutrophils to the fibronectin-coated substrate. Imipramine inhibited neutrophil adhesion and concomitant secretion of proteins, including matrix metalloproteinase 9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL), which modify the extracellular matrix and basement membranes required for cell migration. Imipramine also significantly and selectively blocked the release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, an enzyme that affects the organization of the extracellular matrix by modifying collagen lysine residues. In contrast, imipramine enhanced the release of ROS by neutrophils during adhesion to fibronectin and stimulated apoptosis. The anti-inflammatory effect of imipramine may be associated with the suppression of neutrophil infiltration and their adhesion to nerve tissues by inhibiting the secretion of neutrophils, which provides these processes. [ABSTRACT FROM AUTHOR]

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

Collagen undergoes many types of post-translational modifications (PTMs), including intracellular modifications and extracellular modifications. Among these PTMs, glycosylation of hydroxylysine (Hyl) is the most complicated. Experimental studies demonstrated that this PTM ceases once the collagen triple helix is formed and that Hyl-O-glycosylation modulates collagen fibrillogenesis. However, the underlying atomic-level mechanisms of these phenomena remain unclear. In this study, we first adapted the force field parameters for O-linkages between Hyl and carbohydrates and then investigated the influence of Hyl-O-glycosylation on the structure of type I collagen molecule, by performing comprehensive molecular dynamic simulations in explicit solvent of collagen molecule segment with and without the glycosylation of Hyl. Data analysis demonstrated that (i) collagen triple helices remain in a triple-helical structure upon glycosylation of Hyl; (ii) glycosylation of Hyl modulates the peptide backbone conformation and their solvation environment in the vicinity and (iii) the attached sugars are arranged such that their hydrophilic faces are well exposed to the solvent, while their hydrophobic faces point towards the hydrophobic portions of collagen. The adapted force field parameters for O-linkages between Hyl and carbohydrates will aid future computational studies on proteins with Hyl-O-glycosylation. In addition, this work, for the first time, presents the detailed effect of Hyl-O-glycosylation on the structure of human type I collagen at the atomic level, which may provide insights into the design and manufacture of collagenous biomaterials and the development of biomedical therapies for collagen-related diseases. [ABSTRACT FROM AUTHOR]

The creation of the 9‐fluorenylmethoxycarbonyl (Fmoc) group by the Carpino laboratory facilitated the synthesis of peptides containing acid‐sensitive groups, such as O‐linked glycosides. To fully investigative collagen biochemistry, one needs to assemble peptides that possess glycosylated 5‐hydroxylysine (Hyl). A convenient method for the synthesis of Fmoc‐Hyl(ε‐tert‐butyloxycarbonyl (Boc), O‐tert‐butyldimethylsilyl [TBDMS]) and efficient methods for the synthesis of Fmoc‐Hyl[ε‐Boc,O‐(2,3,4,6‐tetra‐O‐acetyl‐β‐D‐galactopyranosyl)] have been developed. Glycosylated Fmoc‐Hyl derivatives were used to construct a series of types I‐IV collagen‐model triple‐helical peptides (THPs) that incorporated known or proposed receptor binding sites. Glycosylation of Hyl was found to strongly down‐regulate the binding of CD44 and the α3β1 integrin to collagen, while the impact on α2β1 integrin binding was more modest. Molecular modeling of integrin binding indicated that Hyl glycosylation directly impacted the association between the α3β1 integrin metal ion‐dependent adhesion site (MIDAS) and the receptor binding site within type IV collagen. The Fmoc solid‐phase strategy ultimately allowed for chemical biology approaches to be utilized to study tumor cell interactions with glycosylated collagen sequences and document the modulation of receptor interactions by Hyl post‐translational modification. [ABSTRACT FROM AUTHOR]

The analysis of nineteen amino acids found in collagen was optimised using 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) as a derivatisation reagent. The analysis and detection of nineteen AQC-amino acids using fluorescence and mass spectrometry were compared at different mobile phase pH's and column temperatures. The pH range of the mobile phase was set between 2.7 and 6.0 and column temperatures, 15–60 °C. The majority of amino acids produced a mono-derivatised product with AQC, except cystine, lysine and hydroxylysine which were di-derivatised. Hydroxylysine's retention time was affected most by changes in the pH, whilst hydroxyproline's retention time was more affected by column temperature. Hydroxylysine was detected as two diastereomers which were completely resolved. The relative standard deviation of the retention times of AQC-amino acids was less than 1% and the limit of detection (LOD) and limit of quantitation (LOQ) were ranged from (0.05–0.23) µM and (0.07–0.76) µM on fluorescence and (0.02–0.10) µM and (0.06–0.33) µM on mass spectrometry respectively. This method was successfully applied for the quantitation of amino acids in different animal skins. [ABSTRACT FROM AUTHOR]

The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids ( e.g. , HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine ( Met SN Lys/Hyl ) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.

During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.

[Display omitted] • Resistant potato starch (RPS) supplementation decreases serum histamine levels. • RPS does not influence histamine-degrading enzymes. • Relative abundance of histamine-secreting microbes were reduced by RPS. • Markers elevated in intestinal permeability are reduced by RPS. Histamine from our diet or gut microbes can trigger gastrointestinal disturbances, and resistant potato starch (RPS) has previously been shown to alleviate these symptoms while increasing levels of health-associated bacteria such as Akkermansia through unknown mechanisms. Post hoc exploratory metabolomic analysis of serum amino acid, amine, and carnitine metabolites in participants consuming 3.5 g/day RPS or placebo (n = 48) was performed using liquid chromatography-mass spectrometry to determine whether RPS positively influences histamine metabolism and related parameters. Histamine levels were significantly reduced by RPS treatment, but histamine-degrading enzyme products were unaffected by RPS. RPS also reduced histamine-secreting Haemophilus and Lactobacillus. Further, metabolites associated with intestinal permeability, including 5-hydroxylysine, acetylspermidine, and short- and medium-chain carnitines ratios, were significantly reduced by RPS treatment, suggesting decreased serum histamine might be related to enhanced gut barrier function. These metabolomic findings expand the value of supplementing the diet with RPS. [ABSTRACT FROM AUTHOR]

High growth rates and body weight are important traits of young dairy goats that can shorten generation intervals, improve animal performance, and increase economic benefits. In the present study, ninety-nine, 6-month-old, female goats were fed with the same diet and kept under the same management condition. The ten goats with highest average daily gain (ADG, HADG, 135.27 ± 4.59 g/d) and ten goats with lowest ADG (LADG, 87.74 ± 3.13 g/d) were selected to identify the key serum metabolites associated with ADG, and to investigate the relationships of serum metabolome profiles with digestive tract microbiota. The results showed that a total of 125 serum metabolites were significantly different between HADG and LADG. Of these, 43 serum metabolites were significantly higher levels in HADG, including D-ornithine, l-glutamine, L-histidine, carnosine, LysoPC (16:1(9Z)/0:0), DCTP and hydroxylysine, while, 82 serum metabolites were significantly higher levels in LADG, including P-salicylic acid and deoxycholic acid 3-glucuronide. Pathway analysis indicated that these different metabolites were mainly involved in amino acid and lipid metabolism. Furthermore, Spearman's rank correlation analysis revealed that these differential serum metabolites were correlated with ADG and ADG-related bacteria. Notably, serum hydroxylysine and L-histidine could be used as biomarkers for distinguishing HADG and LADG goats, with an accuracy of >92.0%. SIGNIFICANCE: Our study confirms that individual microbiota and metabolic differences contribute to the variations of growth rate in young goats. Some serum metabolites may be useful in improving the growth performance of young goats, which provides directions for developing further nutritional regulation in the goat industry to achieve healthy feeding and efficiency enhancement., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Hydroxylation via COH bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available L-lysine with L-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two L-lysine 3S-hydroxylases and four L-lysine 4Rhydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of L-lysine, yielding (2S,3S)-3- hydroxylysine and (2S,4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant L-lysine hydroxylaseexpressing Escherichia coli cells were used as a biocatalyst for L-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S,3S)-3-hydroxylysine was produced from 600 mM L-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S,4R)-4-hydroxylysine was produced from 300 mM L-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies. IMPORTANCE The present study identified six L-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of L-lysine hydroxylases was relatively narrow, we found that (2S,3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S,5R)-5-hydroxylysine was hydroxylated by both 3S- and 4Rhydroxylases. Moreover, the L-arginine hydroxylase VioC also hydroxylated L-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of L-lysine into (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic L-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses. [ABSTRACT FROM AUTHOR]

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O 2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O 2 -transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Background This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Methods Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. Results It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%–24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. Conclusions The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. General significance The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. [ABSTRACT FROM AUTHOR]

We investigated alternatives to commonly used biomarkers of exercise-induced tissue damage. Over 5 days following two bouts of 100 drop-to-vertical jumps (inter-bout rest period of 3 weeks), myosin heavy chain 1, hydroxylysine (HYL), hydroxyproline (HYP), spermine (SPM) and spermine synthase (SMS) were measured in the serum of 10 participants. HYL significantly increased from 5.92 ± 1.49 ng/mL to 6.48 ± 1.47 ng/mL at 24 h. A similar trend was observed for bout 2, but without reaching significance. SPM significantly increased only after bout 1 from 0.96 ± 0.19 ng/mL at pretest to a peak level of 1.12 ± 0.26 ng/mL at 24 h, while B2 increments remained non-significant. Myosin heavy chain 1, HYP and SMS values remained below the detection limit of the applied enzyme-linked immunosorbent assay (ELISA) kit. Though HYL and SM increased after the intervention, both markers showed a large standard deviation (SD) combined with small increments. Therefore, none of the investigated biomarkers provides a meaningful alternative to commonly used damage markers. [ABSTRACT FROM AUTHOR]

• Improved methods for the reliable and fast analysis of amino acids in food and feed. • Dramatic reduction of analysis time by microwave hydrolysis and UHPLC-MS/MS. • Comparable results to laboratories using ISO, AOAC and regulation (EC) 152/2009 methods. • Determination of hydroxyproline, hydroxylysine, taurine, and ornithine also possible. • High specificity and selectivity due to UHPLC-MS/MS with AQC derivatization. The determination of amino acids in food and feed by chromatography has a long history and is described in several official methods, including standards from ISO, AOAC, and the European Commission (EC) regulation 152/2009. The procedure usually consists of labor- and time-consuming preparation techniques and ion-exchange chromatography with challenging chromatographic conditions. Consequently, several approaches have been published to overcome these drawbacks but the knowledge about their suitability for complex matrices such as food and feed is limited. In this paper, we describe the development of two new methods to determine amino acids in food and feed. These methods involve microwave hydrolysis and reversed-phase UHPLC-MS/MS with pre-column derivatization using 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC). Both methods provide streamlined sample preparations and a dramatic reduction in analysis time while offering a high degree of specificity and selectivity. Selectivity also enabled the simultaneous determination of the more uncommon substances hydroxyproline, hydroxylysine, taurine, ornithine, and γ-amino butyric acid (GABA) along with amino acids typically present in food and feed. The results were all satisfactory with regards to sensitivity, accuracy, precision, and comparability with laboratories that use other methods, for example from ISO, AOAC, or regulation (EC) 152/2009. We therefore concluded that both methods provide a reliable and modern approach to overcome many of the drawbacks that occur with the conventional standard methods. [ABSTRACT FROM AUTHOR]

The Jumonji domain-containing protein 6 (Jmjd6) is a member of the superfamily of non-haem iron(II) and 2-oxoglutarate (2OG)- dependent oxygenases; it plays an important developmental role in higher animals. Jmjd6 was initially assigned a role as the phosphatidylserine receptor responsible for engulfment of apoptotic cells but this nowseems unlikely. Jmjd6 has been shown to be a nuclear localized protein with a JmjC domain comprising a distorted double-stranded β-helical structure characteristic of the 2OG-dependent oxygenases. Jmjd6 was subsequently assigned a role in catalysing N-methyl-arginine residue demethylation on the N-terminus of the human histones H3 and H4; however, this function is also subject to conflicting reports. Jmjd6 does catalyse 2OG-dependent C-5 hydroxylation of lysine residues in mRNA splicing-regulatory proteins and histones; there is also accumulating evidence that Jmjd6 plays a role in splicing (potentially in an iron- and oxygen-dependent manner) as well as in other processes regulating gene expression, including transcriptional pause release. Moreover, a link with tumour progression has been suggested. In the present review we look at biochemical, structural and cellular work on Jmjd6, highlighting areas of controversy and consensus. [ABSTRACT FROM AUTHOR]

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5-hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2-aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne-containing benzimidate to form a 2-oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide-substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne-labeled oxazoline form. The Koh-labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation. [ABSTRACT FROM AUTHOR]

This review article gives a survey of principal pathways that lead to the biosynthesis of the modified principal proteinogenic amino acids, i.e. cystine, 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, and O-phosphoserine. Except the proteinogenic amino acids, peptides and proteins often contain several unusual amino acids arising by specific modifications (e.g. oxidation or esterification) of amino acid residues present in the already synthesised polypeptide chain. The post-translational products include, e.g., the oxidation of the thiol groups of two cysteine residues to form a disulfide bridge (cystine), thus allowing cross-linking of polypeptide chains; the hydroxylation of proline to 4-hydroxyproline and of lysine to 5-hydroxylysine; N-methylation of histidine to 3-methylhistidine; and the phosphorylation of serine to O-phosphoserine. There also exist several other modified proteinogenic amino acids that are of minor significance to foods.