Journal: Nutrients

Article Title: Evaluation of the Relationship Between Orexin A, Peptide YY, AgRP, and POMC Levels and Sleep Disorders in Children with Malnutrition

doi: 10.3390/nu18030377

Figure Lengend Snippet: Box plots showing the distribution of the orexin A, proopiomelanocortin (POMC), agouti-related protein (AgRP), and peptide yy (PYY) levels in the malnourished children and typically developing (TD) healthy controls. The Mann–Whitney U test was used to compare the peptide levels between the two groups.

Article Snippet: Human POMC, orexin A, AgRP, and PYY enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Korain Biotech Co., Ltd., Shanghai, China) were used to measure the plasma peptide levels.

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Molecular evolution of the proopiomelanocortin system in Barn owl species

doi: 10.1371/journal.pone.0231163

Figure Lengend Snippet: (A) Summary on the hierarchical processing of POMC by proprotein convertases PC1/3 and PC2 [ , – ]. (B) Schematic representation of Tyto alba alba (TA) / Strix aluco (SA) POMC with putative dibasic cleavage sites indicated by scissors. Note the additional RR motif within the Joining peptide in TA. The amino acid (aa) sequences of β-MSH for both owl species are indicated above the polypeptide precursor. (C) Synthetic β-MSH peptide variants used to assess receptor signaling. The naturally occurring β-MSH variants in SA and TA are under-laid in gray and are designated as wild-type (wt), with differing aa depicted by enlarged letters. In artificial variants, aa in agreement with TA or SA wt forms are in red and blue, respectively. Wt TA β-MSH variants carry proline in position 9 (P9) as part of the P FRW receptor-binding motif (yellow), whereas β-MSH of SA displays histidine in position 9 (H9). (D-F) Receptor-signaling of β-MSH variants. Recombinant TA MC3R or MC4R were transiently expressed in HEK293T cells, followed by exposure to synthetic β-MSH peptides at the indicated concentrations. Receptor signaling activity was assessed by monitoring cellular cAMP levels via Glo-sensor in a luminescence assay as detailed in Materials and Methods. Data are means + SEM, n = 3. Dose-response curves were calculated by non-linear regression analysis. (G, H) TA β-MSH P9 cannot compete with the hypothetic TA β-MSH H9 form in receptor signaling, neither on MC3R (G) nor MC4R (H).

Article Snippet: SA β-MSH H9: AGGSYRMR HFRW HAPLKD (GenScript USA Inc., Piscataway, New Jersey, USA); SA β-MSH P9: AGGSYRMR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9: AGGSHRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH H9: AGGSHRVR HFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, Y5: AGGSYRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, M7: AGGSHRMR PFRW HAPLKD (GenScript USA Inc.); SA / TA γ2-MSH: YVMSHFRWNKFG, C-terminus amidated (GenScript USA Inc.); SA γ3-MSH 3 S: YVMSHFRWNKFGRRNSSSGGGGGH (GenScript USA Inc.); TA γ3-MSH 18S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSGGH (GenScript USA Inc.); TA γ3-MSH 7 S: YVMSHFRWNKFGRRNSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 11 S: YVMSHFRWNKFGRRNSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 13 S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 19S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 5 S: YVMSHFRWNKFGRRNSSSSSGGH (SynPeptide CO., LTD, Shanghai, China); TA γ3-MSH 10 S: YVMSHFRWNKFGRRNSSSSSSSSSSGGH (SynPeptide CO., LTD); human β-MSH H9 (wt): DEGPYRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH P9: DEGPYRMEPFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH C5: DEGPCRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH L15: DEGPYRMEHFRWGSLPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ2-MSH: YVMGHFRWDRFG-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH (wt): YVMGHFRWDRFGRRNSSSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGAGQ (SynPeptide CO., LTD, Shanghai, China).

Techniques: Binding Assay, Recombinant, Activity Assay, Luminescence Assay

Journal: PLoS ONE

Article Title: Molecular evolution of the proopiomelanocortin system in Barn owl species

doi: 10.1371/journal.pone.0231163

Figure Lengend Snippet: Synthetic peptides of TA and SA γ2-MSH, SA γ3-MSH with 3 serine residues, or TA γ3-MSH with 5, 7, 10, 11, 13, 18 and 19 serine residues were diluted in human plasma (A), or water (C) to final concentrations of 2 μM and incubated at 37°C. Incubations for all γ-MSH peptides were performed in triplicate. Aliquots were removed after different time points for up to 68 h, and the remaining cAMP signaling activity was simultaneously determined on TA MC3R and Glo-sensor transfected HEK293T cells by luminescence assay. Measured maximal luminescence values were normalized to values at time point zero and half-lives were determined from one-phase decay curves (A, B). γ2-MSH, γ3-MSH and their breakdown products in blood plasma did not induce any signal on non-MC3R, but Glo-sensor transfected cells . No loss in signaling activity was observed in water up to 68 h neither for γ2- nor for γ3-MSH, therefore excluding non-enzymatic decay or signaling loss over time due to peptide aggregation (C). (D, E) The inactive TA β-MSH P9 with its dysfunctional receptor-binding site PFRW does not act as a competitive antagonist for γ-MSH signaling on MC3R or MC4R, neither for γ2-MSH nor for γ3-MSH 10 serine variant. (F-I) Dose response curves for human MC3R (F, H) and MC4R signaling (G, I) of synthetic human β-MSH H9 (wild-type, Y5, P15), and mutant β-MSH P9, β-MSH Y5C and β-MSH P15L peptides. Data are means + SEM, n = 3. (J-K) Signaling of synthetic human non-glycosylated γ2- and γ3-MSH peptides (wild-type or with one or two SSG extensions in the serine-rich tail) on MC3R (J) and MC4R (K). Data are means + SEM, n = 3. EC50 were determined from dose response curves established after non-linear regression curve fit for the different human β- and γ-MSH peptides as described (Figs and ) and were normalized (EC50 n ) to the EC50 obtained for wild-type human β- (F-I) and γ2-MSH peptides (J, K). (L, M) Plasma stability of human γ2-MSH or γ3-MSH peptides (wild-type or γ3-MSH SSG or γ3-MSH SSG SSG) was assessed on human MC3R as described above for owl γ-MSH peptides on TA MC3R (A). Background signaling of residual human γ3-MSH peptides was established on non-MC3R, but Glo-sensor transfected cells (L, M).

Article Snippet: SA β-MSH H9: AGGSYRMR HFRW HAPLKD (GenScript USA Inc., Piscataway, New Jersey, USA); SA β-MSH P9: AGGSYRMR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9: AGGSHRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH H9: AGGSHRVR HFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, Y5: AGGSYRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, M7: AGGSHRMR PFRW HAPLKD (GenScript USA Inc.); SA / TA γ2-MSH: YVMSHFRWNKFG, C-terminus amidated (GenScript USA Inc.); SA γ3-MSH 3 S: YVMSHFRWNKFGRRNSSSGGGGGH (GenScript USA Inc.); TA γ3-MSH 18S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSGGH (GenScript USA Inc.); TA γ3-MSH 7 S: YVMSHFRWNKFGRRNSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 11 S: YVMSHFRWNKFGRRNSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 13 S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 19S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 5 S: YVMSHFRWNKFGRRNSSSSSGGH (SynPeptide CO., LTD, Shanghai, China); TA γ3-MSH 10 S: YVMSHFRWNKFGRRNSSSSSSSSSSGGH (SynPeptide CO., LTD); human β-MSH H9 (wt): DEGPYRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH P9: DEGPYRMEPFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH C5: DEGPCRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH L15: DEGPYRMEHFRWGSLPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ2-MSH: YVMGHFRWDRFG-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH (wt): YVMGHFRWDRFGRRNSSSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGAGQ (SynPeptide CO., LTD, Shanghai, China).

Techniques: Incubation, Activity Assay, Transfection, Luminescence Assay, Binding Assay, Variant Assay, Mutagenesis

Journal: PLoS ONE

Article Title: Molecular evolution of the proopiomelanocortin system in Barn owl species

doi: 10.1371/journal.pone.0231163

Figure Lengend Snippet: (A) Schematic representation of recombinant double-tagged (HA, V5) SA / TA POMC precursor with dibasic cleavage sites 1 to 10, required for production of the peptide hormones γ2-MSH, γ3-MSH, ACTH, α-MSH, CLIP, β-LPH, γ-LPH, β-MSH, and β-end. The N-glycan at the asparagine residue within γ3-MSH is depicted as black pentagon. The polymorphisms in γ3-MSH and β-MSH are shown above the POMC precursor. N-terminal cleavage fragments up to sites 1 through 10 are listed below the precursor, and corresponding bands in the below Western blots are labeled by the according numbers in red. Fragments 3u, 5u, 6u likely correspond to under-glycosylated forms of fragments 3, 5 and 6, respectively. The full length POMC precursor is labeled with PP. The indicated molecular masses of fragments were calculated as detailed in Supplemental Information and . (B) Cleavage of TA / SA POMC variants by TA PC1/3 and PC2 (with 7B2) in HEK293T cells. Cell lysates and supernatants were harvested 56 h post transfection and N-terminal POMC fragments were detected in Western blot with an anti-HA antibody. Note that the PC1/3-specific fragment resulting from cleavage at site 2 (red arrow) is present in wt SA POMC with 3 serine residues, but not in TA POMC with 10 or 18 serine residues (asterisks indicating missing fragments produced by the respective PC). Reducing the number of serine residues in TA POMC to 3 leads to re-appearance of band 2 (green arrow), and increasing the number of serine residues in SA POMC to 18 to its disappearance. Identified PC1/3 and PC2 specific cleavage sites in SA and TA POMC are indicated in the bottom schematic below the scissors.

Article Snippet: SA β-MSH H9: AGGSYRMR HFRW HAPLKD (GenScript USA Inc., Piscataway, New Jersey, USA); SA β-MSH P9: AGGSYRMR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9: AGGSHRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH H9: AGGSHRVR HFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, Y5: AGGSYRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, M7: AGGSHRMR PFRW HAPLKD (GenScript USA Inc.); SA / TA γ2-MSH: YVMSHFRWNKFG, C-terminus amidated (GenScript USA Inc.); SA γ3-MSH 3 S: YVMSHFRWNKFGRRNSSSGGGGGH (GenScript USA Inc.); TA γ3-MSH 18S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSGGH (GenScript USA Inc.); TA γ3-MSH 7 S: YVMSHFRWNKFGRRNSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 11 S: YVMSHFRWNKFGRRNSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 13 S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 19S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 5 S: YVMSHFRWNKFGRRNSSSSSGGH (SynPeptide CO., LTD, Shanghai, China); TA γ3-MSH 10 S: YVMSHFRWNKFGRRNSSSSSSSSSSGGH (SynPeptide CO., LTD); human β-MSH H9 (wt): DEGPYRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH P9: DEGPYRMEPFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH C5: DEGPCRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH L15: DEGPYRMEHFRWGSLPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ2-MSH: YVMGHFRWDRFG-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH (wt): YVMGHFRWDRFGRRNSSSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGAGQ (SynPeptide CO., LTD, Shanghai, China).

Techniques: Recombinant, Western Blot, Labeling, Transfection, Produced

Journal: PLoS ONE

Article Title: Molecular evolution of the proopiomelanocortin system in Barn owl species

doi: 10.1371/journal.pone.0231163

Figure Lengend Snippet: (A) Phenotypes associated with polymorphisms in β- and γ-MSH. (B, C) Working model: POMC and MC3R-expressing neurons in the arcuate nucleus of the hypothalamus projecting to MC4R expressing neurons of the paraventricular nucleus are shown. Anticipated MSH signaling scenarios are depicted for the combination of intact β-MSH H9 and γ3-MSH with < 7 serine residues (5 S) as in ancient clades of Tydonidae (B), and for dysfunctional β-MSH P9 and γ3-MSH with > 7 serine residues (10 S) as found in the barn owl group (C). The extension of serine residues in the POMC γ3-MSH locus to ≥7 increases the production of γ3-MSH at the cost of γ2-MSH, as exemplified by the γ3-MSH 10 S variant in the barn owl group (C). Since the MC3R signaling activity of γ3-MSH is reduced by approximately one order of magnitude compared to γ2-MSH, the auto-inhibitory γ-MSH activity on POMC expressing neurons of the arcuate nucleus is strongly decreased in C. As a consequence, the release of POMC derived MSH peptides is prolonged. The lack of β-MSH P9 signaling on post-synaptic MC4R in the paraventricular nucleus can therefore be compensated by increased amounts of α-MSH in the synaptic cleft. The elevated cAMP levels in neurons of the paraventricular nucleus resulting from MC4R activation can therefore be maintained and an energy imbalance prevented, despite dysfunctional β-MSH P9.

Article Snippet: SA β-MSH H9: AGGSYRMR HFRW HAPLKD (GenScript USA Inc., Piscataway, New Jersey, USA); SA β-MSH P9: AGGSYRMR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9: AGGSHRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH H9: AGGSHRVR HFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, Y5: AGGSYRVR PFRW HAPLKD (GenScript USA Inc.); TA β-MSH P9, M7: AGGSHRMR PFRW HAPLKD (GenScript USA Inc.); SA / TA γ2-MSH: YVMSHFRWNKFG, C-terminus amidated (GenScript USA Inc.); SA γ3-MSH 3 S: YVMSHFRWNKFGRRNSSSGGGGGH (GenScript USA Inc.); TA γ3-MSH 18S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSGGH (GenScript USA Inc.); TA γ3-MSH 7 S: YVMSHFRWNKFGRRNSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 11 S: YVMSHFRWNKFGRRNSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 13 S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd, Shanghai, China); TA γ3-MSH 19S: YVMSHFRWNKFGRRNSSSSSSSSSSSSSSSSSSSGGH (Top-peptide Co., Ltd); TA γ3-MSH 5 S: YVMSHFRWNKFGRRNSSSSSGGH (SynPeptide CO., LTD, Shanghai, China); TA γ3-MSH 10 S: YVMSHFRWNKFGRRNSSSSSSSSSSGGH (SynPeptide CO., LTD); human β-MSH H9 (wt): DEGPYRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH P9: DEGPYRMEPFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH C5: DEGPCRMEHFRWGSPPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human β-MSH L15: DEGPYRMEHFRWGSLPKD-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ2-MSH: YVMGHFRWDRFG-NH2 (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH (wt): YVMGHFRWDRFGRRNSSSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGSSGAGQ (Top-peptide Co., Ltd, Shanghai, China); human γ3-MSH SSG: YVMGHFRWDRFGRRNSSSSGSSGSSGAGQ (SynPeptide CO., LTD, Shanghai, China).

Techniques: Expressing, Variant Assay, Activity Assay, Derivative Assay, Activation Assay