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Biosynthesis and physiological functions of N-acyl amino acids
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

N-acyl amino acids are lipid signalling molecules that have recently been identified in biological systems. These lipids are structurally related to the endocannabinoids, although they do not activate cannabinoid receptors. In 2001, N-arachidonoyl glycine was the first signalling lipid in this group to be identified in bovine and rat brain and since then, about 50 novel N-acyl amino acids have been identified in mammalian systems. These N-acyl amino acids are involved in regulating pain processes, are anti-inflammatory and regulate body temperature, but the metabolic pathways for production and metabolism remain poorly understood.

This thesis focussed on the identification of pathways for production and regulation of N-acyl amino acids, in particular N-acyl glycines, and in identifying physiological functions for N-acyl amino acids (particularly N-acyl taurines). Our results identified an enzymatic pathway for production of N-acyl glycines in human and we identified that the human glycine N-acyltransferase-like 2 (hGLYATL2) conjugates (amidates) medium- and long-chain, saturated and unsaturated acyl-CoAs with glycine, to produce N-acyl glycines, with the preferential production of N-oleoyl glycine. Furthermore, we have characterized two other members of the gene family of glycine N-acyltransferases (GLYATs) in human, the hGLYATL1 and hGLYATL3 that may be involved in the production of N-acyl amino acids.

As N-acyl glycines are bioactive signalling molecules, it is likely their production requires a rapid on/off switch. The post-translational modification of proteins can result in enzyme regulation, without the need for transcriptional regulation. We have identified that hGLYATL2 is regulated by acetylation/deacetylation on lysine 19, and using mutation analysis, we show that deacetylation of lysine 19 is important for full enzyme activity.

The physiological functions of N-acyl amino acids are not well studied to date. In this thesis, we have identified that N-arachidonoyl taurine and N-oleoyl taurine trigger insulin secretion by increasing the calcium flux in pancreatic b-cells via the activation of transient receptor potential vanilloid subfamily 1 (TRPV1).

This work on N-acyl amino acids has led us to identify new pathways and physiological functions for these lipid signalling molecules, which advances our knowledge of the importance of these lipids in mammalian systems.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University , 2012. , 62 p.
National Category
Cell Biology Biochemistry and Molecular Biology Genetics
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-75766ISBN: 978-91-7447-523-4 (print)OAI: oai:DiVA.org:su-75766DiVA: diva2:523865
Public defence
2012-06-01, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of doctoral defence the following papers were unpublished and had a status as follows:Paper 2: Accepted; Paper 3: Manuscript; Paper 4; Manuscript

Available from: 2012-05-10 Created: 2012-04-26 Last updated: 2013-04-09Bibliographically approved
List of papers
1. Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans.
Open this publication in new window or tab >>Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans.
2010 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 8, 2795-2803 p.Article in journal (Refereed) Published
Abstract [en]

The discovery of glycine conjugates of long-chain fatty acids (N-acyl glycines) in the brain and other non-neuronal tissues has led to the identification of an emerging class of bioactive lipids. The biological activities of N-acyl glycines include antinociceptive, anti-inflammatory and antiproliferative effects, and activation of G-protein-coupled receptors. However, despite the fact that N-acyl glycines are emerging as a distinct lipid signaling family, pathways for their production are not fully elucidated. Here we report on the characterization of human glycine N-acyltransferase-like 2 (hGLYATL2), a member of a gene family of 4 putative glycine conjugating enzymes, and show that it synthesizes various N-acyl glycines. Recombinantly expressed hGLYATL2 efficiently conjugated oleoyl-CoA, arachidonoyl-CoA, and other medium- and long-chain acyl-CoAs to glycine. The enzyme was specific for glycine as an acceptor molecule, and preferentially produced N-oleoyl glycine. The hGLYATL2 enzyme is localized to the endoplasmic reticulum, and the mRNA shows highest expression in salivary gland and trachea, but is also detected in spinal cord and skin fibroblasts. The expression pattern and the identification of high levels of N-acyl glycines in skin and lung may indicate a role for N-acyl glycines in barrier function/immune response and the potential role of hGLYATL2 in this regard is discussed.

Keyword
N-oleoyl glycine, N-arachidonoyl glycine, endocannabinoids, N-acyl amino acids, endoplasmic reticulum
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Molecular Cellbiology; Molecular Genetics
Identifiers
urn:nbn:se:su:diva-75625 (URN)10.1096/fj.09-148551 (DOI)
Funder
Swedish Research Council
Available from: 2012-04-23 Created: 2012-04-23 Last updated: 2017-12-07Bibliographically approved
2. Reversible lysine acetylation regulates the activity of human glycine n-acyltransferase-like 2 (hGLYATL2): Implications for production of glycine-conjugated signalling molecules
Open this publication in new window or tab >>Reversible lysine acetylation regulates the activity of human glycine n-acyltransferase-like 2 (hGLYATL2): Implications for production of glycine-conjugated signalling molecules
Show others...
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 20, 16158-16167 p.Article in journal (Refereed) Published
Abstract [en]

Lysine acetylation is a major post-translational modification of proteins, and regulates many physiological processes such as metabolism, cell migration, ageing and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim et al, (2006) Mol. Cell. 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19 (K19). Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 (K19) in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that K19 is not acetylated in wild-type hGLYATL2, indicating that K19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signalling molecules that regulate functions like the perception of pain, body temperature, and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulate the enzyme activity, thus linking post-translational modification of proteins with the production of biological signalling molecules, the N-acyl glycines.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Molecular Biology; Cell Biology
Identifiers
urn:nbn:se:su:diva-75627 (URN)10.1074/jbc.M112.347260 (DOI)000304030900008 ()
Available from: 2012-04-26 Created: 2012-04-23 Last updated: 2017-12-07Bibliographically approved
3. Molecular characterization of two members of the glycine N-acyltransferase gene family in human: glycine N-acyl transferase-like 1 (GLYATL1) and glycine N-acyltransferase-like 3 (GLYATL3).
Open this publication in new window or tab >>Molecular characterization of two members of the glycine N-acyltransferase gene family in human: glycine N-acyl transferase-like 1 (GLYATL1) and glycine N-acyltransferase-like 3 (GLYATL3).
(English)Manuscript (preprint) (Other academic)
Abstract [en]

N-acyl amino acids are a group of endogenous lipid mediators that regulate a variety of cellular physiological functions. The discovery of N-acyl amino acids in many biological systems has allowed research to focus on their functions as well as pathways for production of these signalling lipids.

We have previously identified that human glycine N-acyltransferase-like 2 (hGLYATL2) is involved in the enzymatic formation of N-acyl glycines. hGLYATL2 is localized in a gene cluster with other glycine N-acyltransferase genes. Here, we have characterized human glycine N-acyltransferase-like 1 (hGLYATL1) and human glycine N-acyltransferase-like 3 (hGLYATL3), which are members of this gene family. Our results show that hGLYATL1 is localized to the endoplasmic reticulum (ER) but the intracellular localization of hGLYATL3 remains to be determined. The hGLYATL1 mRNA shows highest expression in liver and kidney, whereas mRNA of hGLYATL3 is expressed in pancreas and liver. Using bioinformatics we determined the overall three-dimensional (3D) structures of hGLYATL1 and hGLYATL3 enzymes, with predicted binding site residues.

In summary, we have characterized novel members of glycine N-acyltransferases that may be involved in the production of lipid signalling molecules, in particular N-acyl glycines.

National Category
Biochemistry and Molecular Biology
Research subject
Genetics; Cell Biology; Biochemistry; Biochemistry towards Bioinformatics
Identifiers
urn:nbn:se:su:diva-75764 (URN)
Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2012-05-02Bibliographically approved
4. N-acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic b-cells
Open this publication in new window or tab >>N-acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic b-cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Pancreatic b-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signalling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic b-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic b-cell lines as determined by insulin release assay and immunofluorescence (p<0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Cell Biology
Identifiers
urn:nbn:se:su:diva-75760 (URN)
Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2012-05-02Bibliographically approved

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