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Functional characterization in vitro of twelve naturally occurring variants of the human pancreatic polypeptide receptor NPY4R
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Uppsala University.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-5578-7996
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.ORCID iD: 0000-0002-2913-514X
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.ORCID iD: 0000-0003-0459-3491
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2019 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, article id 101933Article in journal (Refereed) Published
Abstract [en]

Obesity has become a global health problem and therefore understanding of the mechanisms regulating hunger and satiety is of utmost importance for the development of new treatment strategies. The Y4 receptor, encoded by the NPY4R gene, and its ligand pancreatic polypeptide (PP) have been reported to mediate a satiety signal. Multiple genetic studies have reported an association between NPY4R copy number and body weight. The gene also displays several SNP variants, many of which lead to amino acid differences, making it interesting to study. We have investigated the functional properties of 12 naturally occurring amino acid sequence variants of the Y4 and interpret the results in relation to sequence conservation and our structural model of the human Y4 receptor protein. Three receptor variants, Cys201(ECL2)Tyr, Val271(6.41)Leu and Asn318(7.49)Asp, were found to completely lose functional response, measured as inositol phosphate turnover, while retaining membrane expression. They display high sequence conservation and have important roles in the receptor structure. For two receptor variants the potency of PP was significantly decreased, Cys34(NT)Ser (EC50 = 2.9 nM, p < .001) and Val135(3.46)Met (EC50 = 3.0 nM, p < .01), compared to wild-type Y4 (EC50 = 0.68 nM). Cys34 forms a disulphide bond with Cys298, linking the N-terminal part to ECL3. The Val135(3.46)Met variant has an amino acid replacement located in the TM3 helix, one helix turn above the highly conserved ERH motif. This position has influence on the network of residues involved in receptor activation and subsequent inactivation. Sequence conservation and the structural model are consistent with these results. The remaining seven positions had no significant effect on the receptor's functional response compared to wild-type Y4. These positions display more variation during evolution. Understanding of the interactions between the Y4 receptor and its native PP agonist and the effects of amino acid variation on its functional response will hopefully lead to future therapeutic possibilities.

Place, publisher, year, edition, pages
CHURCHILL LIVINGSTONE , 2019. article id 101933
Keywords [en]
Y4, SNP, Mutagenesis, Functional pharmacology, Structural modelling
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-356572DOI: 10.1016/j.npep.2019.05.004PubMedID: 31230758OAI: oai:DiVA.org:uu-356572DiVA, id: diva2:1236211
Funder
Swedish Research Council, K2013-55 x -22189-01-2Available from: 2018-08-01 Created: 2018-08-01 Last updated: 2019-10-23Bibliographically approved
In thesis
1. The human pancreatic polypeptide receptor Y4: Genetic and functional variation
Open this publication in new window or tab >>The human pancreatic polypeptide receptor Y4: Genetic and functional variation
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Humans are evolutionarily adapted to an environment where food is scarce, but today many live in a world of food abundance. Paired with low physical activity, this may lead to weight gain and obesity. Efficient anti-obesity treatments require understanding of the mechanisms that control hunger, satiety, energy metabolism and body weight. This thesis investigates possible genetic and physiological mechanisms behind these processes.

Genetic correlation between body-mass index (BMI) and a highly polymorphic region on chromosome 10 was analysed with regard to single nucleotide polymorphisms (SNPs) and gene copy number variation (CNV). This region contains the gene NPY4R encoding the pancreatic polypeptide (PP) receptor Y4, which has been reported to reduce appetite.

The results show that the NPY4R gene was duplicated before the divergence of modern humans from the Neanderthals and the Denisovans (approximately to 400,000–800,000 years ago). The CNV of the NPY4R gene region was investigated by read depth analysis based on genome sequences and droplet digital PCR (ddPCR). The read depth results revealed a CNV range of 3-7 copies per genome, while the ddPCR results demonstrated a range of 2–11. Most humans have a total of 4–5 copies, in contrast to the two copies presumed by previous studies.

Investigation of an association between the NPY4R CNV and body mass index (BMI) led to interesting and ambiguous results. A study of 558 Swedish individuals with a wide range of BMI suggested, surprisingly, a positive correlation between NPY4R copy number and BMI for women. On the other hand, a study of 1009 individuals from Northern Sweden found no correlation between BMI and NPY4R copy number. These diverging findings may be due to geographical variation or lack of power in one of these studies.

Twelve naturally-occurring amino acid variants of the Y4 receptor were investigated pharmacologically in cell culture. Three of these showed no functional response, which may be explained by altered conformation of the receptors. For two receptor variants PP had a significantly decreased potency. A 3D model of the Y4 receptor was generated based on the crystal structure of the human Y1 receptor. The functional responses of the Y4 variants agree well with the 3D model and with the degree of evolutionary conservation of the positions.

In conclusion, these studies reveal unexpectedly large CNV as well as extensive SNP for the NPY4R gene and a possible correlation with BMI that may be due to the differing responses of the naturally occurring receptor variants.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 53
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1479
Keywords
NPY4R, Y4, obesity, CNV
National Category
Biochemistry and Molecular Biology Genetics
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-356573 (URN)978-91-513-0389-5 (ISBN)
Public defence
2018-09-18, C2:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-08-24 Created: 2018-08-01 Last updated: 2018-09-07
2. Computational prediction of ligand binding in peptide G-protein coupled receptors
Open this publication in new window or tab >>Computational prediction of ligand binding in peptide G-protein coupled receptors
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

G-protein coupled receptors (GPCRs) are a superfamily of membrane receptors involved in a wide variety of biological processes, and their malfunction is associated with many diseases. Consequently, GPCRs are targeted by one-third of the drugs on the market, and constitute the focus of active public and private research in the search of more effective drugs. The GPCR families that are activated by endogenous peptides are particularly challenging for the drug design process, which in this case contemplates peptides, peptidomimetics and small molecules, as selective activators (agonists) or blockers (antagonists) of the particular receptor subtype of interest. This process benefits of a detailed understanding of how known ligands bind to the receptors. Homology modelling, molecular dynamics (MD) and free energy perturbation (FEP) are computational methods used to predict binding modes and binding affinities. In this thesis, these techniques are applied (and even further developed) in combination with novel experimental data provided by our collaborators, in order to elucidate the molecular determinants of endogenous peptide ligands, analogues and mimetics to two families of peptide-binding receptors: the neuropeptide Y (NPY) and the Angiotensin II receptors.

The NPY signaling system is responsible for the regulation of food intake and its malfunction is connected to obesity, a risk factor for diseases such as diabetes and cancer. In this thesis, we focused on the elucidation of the binding mode of endogenous peptide ligands and studied the structural effect of receptor mutants, with the aim of helping in future drug design on the Y2 receptor subtype, as well as understanding the effect of receptor polymorphisms on the Y4 subtype. We further used this system to refine and test our computational protocol for the prediction of binding free energies, by characterizing the binding mode of a peptidomimetic antagonist to the Y1 receptor.

The AT2 receptor is another interesting drug target, as its activation by the Angiotensin II peptide elicits responses that counterbalance the hypertensive effects caused by activation of the AT1 receptor by the same ligand. Moreover, AT2 is upregulated in events of tissue damage. We characterized the chemical evolution of peptide and peptidomimetic agonists at this receptor, with the aim to identify a set of pharmacophoric points and key interactions with AT2. The outcome of this study allowed the establishment of a clear explanation of structure-activity relationships, and will be the starting point for further ligand-design efforts at this receptor.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1875
Keywords
GPCR, neuropeptide Y, angiotensin II receptor, molecular dynamics, free energy perturbation, homology modelling, computer simulations, peptide binding, peptidomimetics, binding free energy.
National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-395761 (URN)978-91-513-0796-1 (ISBN)
Public defence
2019-12-12, B22, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-21 Created: 2019-10-23 Last updated: 2019-11-21

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Vasile, SilvanaXu, BoGutiérrez-de-Terán, HugoLarhammar, Dan

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