Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide

Haylor, J.; Parker, Ernest T.; Risbridger, Gail P.; Beale, D.; Brown, Barry L.; Dobson, P. R. M.; Clarke, I. J.; Hart, John E.

doi:10.1016/j.regpep.2008.10.011

Cited by 8 publications

(21 citation statements)

References 28 publications

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“…Rabbits at a supplier (Eurogentec, Liège, Belgium) were injected subcutaneously with the antigen (400 μg protein) in PBS mixed with an equal volume of complete Freund’s adjuvant followed by subsequent injections at monthly intervals in Freund’s incomplete adjuvant. An ELISA dilution curve for the rabbit polyclonal antiserum (ER88) has been provided elsewhere ( Haylor et al, 2009 ). A polyclonal antiserum (G530) was raised in goat (Monash University, Victoria, Australia) in a similar manner ( Hart, 2013 ) and an antibody ELISA dilution curve obtained using the same standard methodology ( Haylor et al, 2009 ).…”

Section: Methodsmentioning

confidence: 99%

“…An ELISA dilution curve for the rabbit polyclonal antiserum (ER88) has been provided elsewhere ( Haylor et al, 2009 ). A polyclonal antiserum (G530) was raised in goat (Monash University, Victoria, Australia) in a similar manner ( Hart, 2013 ) and an antibody ELISA dilution curve obtained using the same standard methodology ( Haylor et al, 2009 ). Titre was also established via IHC, using rat and ovine median eminence tissue (basal hypothalamus), and the ability to block antibody with EPL001 was determined on histological sections at 0.5 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…The sequence was synthesized to provide a 14mer peptide, which was given the proprietary designation EPL001. This proved to have dose-dependent tissue-reducing properties in an in vivo model system involving compensatory renal growth in the unilaterally nephrectomised rat ( Haylor et al, 2009 ). Conversely, an antibody to EPL001 exaggerated compensatory renal growth, suggesting that an endogenous inhibitor had been neutralized.…”

Section: Introductionmentioning

confidence: 99%

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Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Hart¹,

Clarke

Risbridger

et al. 2017

PeerJ

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In the context of a hunt for a postulated hormone that is tissue-mass inhibiting and reproductively associated, there is described probable relatedness to a granin protein. A 7–8 kDa polypeptide candidate (gels/MS) appeared in a bioassay-guided fractionation campaign involving sheep plasma. An N-terminal sequence of 14 amino acids was obtained for the polypeptide by Edman degradation. Bioinformatics and molecular biology failed to illuminate any ovine or non-ovine protein which might relate to this sequence. The N-terminal sequence was synthesized as the 14mer EPL001 peptide and surprisingly found to be inhibitory in an assay in vivo of compensatory renal growth in the rat and modulatory of nematode fecundity, in line with the inhibitory hormone hypothesis. Antibodies were raised to EPL001 and their deployment upheld the hypothesis that the EPL001 amino acid sequence is meaningful and relevant, notwithstanding bioinformatic obscurity. Immunohistochemistry (IHC) in sheep, rodents and humans yielded staining of seeming endocrine relevance (e.g. hypothalamus, gonads and neuroendocrine cells in diverse tissues), with apparent upregulation in certain human tumours (e.g. pheochromocytoma). Discrete IHC staining in Drosophila melanogaster embryo brain was seen in glia and in neuroendocrine cells, with staining likely in the corpus cardiacum. The search for the endogenous antigen involved immunoprecipitation (IP) followed by liquid chromatography and mass spectrometry (LC–MS). Feedstocks were PC12 conditioned medium and aqueous extract of rat hypothalamus—both of which had anti-proliferative and pro-apoptotic effects in an assay in vitro involving rat bone marrow cells, which inhibition was subject to prior immunodepletion with an anti-EPL001 antibody—together with fruit fly embryo material. It is concluded that the mammalian antigen is likely secretogranin II (SgII) related. The originally seen 7–8 kDa polypeptide is suggested to be a new proteoform of secretogranin II of ∼70 residues, SgII-70, with the anti-EPL001 antibody seeing a discontinuous epitope. The fly antigen is probably Q9W2X8 (UniProt), an uncharacterised protein newly disclosed as a granin and provisionally dubbed macrogranin I (MgI). SgII and Q9W2X8 merit further investigation in the context of tissue-mass inhibition.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Hart¹,

Clarke

Risbridger

et al. 2017

PeerJ

View full text Add to dashboard Cite

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“…The peptide EPL001 arose from a hormone discovery programme directed at finding an endogenous tissue-mass inhibitor in mammals (Haylor et al, 2009;Hart, 2014). An N-terminal sequence of 14 amino acids was obtained from ovine material, but bioinformatics failed to identify the parent molecule.…”

Section: Introductionmentioning

confidence: 99%

“…A 14mer peptide corresponding to this N-terminal sequence was synthesized. It was designated EPL001 and shown to have biological activity: inhibition of compensatory renal growth was observed in the rat following unilateral nephrectomy (Haylor et al, 2009). As this system is associated with IGF1 and the related DAF2 pathway is known to be important in longevity and reproduction in C. elegans (Baumeister et al, 2006), it was of interest to test EPL001 in this nematode in terms of lifespan and fecundity (Davies and Hart, 2008).…”

Section: Introductionmentioning

confidence: 99%

Reproduction potentiated in nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) by adding a synthetic peptide to their aqueous environment

Davies

Zimmerman

Dudley

et al. 2015

Journal of Experimental Biology

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Ambient exposure to a short synthetic peptide has enhanced fecundity (number of offspring) in invertebrates and vertebrates, ostensibly by disinhibiting reproduction. In separate experiments, nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) were exposed via their aqueous environment to a dissolved synthetic hexamer (6mer) peptide, IEPVFT (EPL036), at a concentration of. In the case of the worms, peptide was added to their aqueous buffer daily throughout the experiment (14 days); for the guppies, peptide administration was on the first 15 alternate days in a 50 week experiment. Fecundity rose by 79% among the worms. The number of descendants of the treated guppies was more than four times that of controls by week 26 (103 versus 25, including 72 juveniles versus 6), with 15.4% more estimated biomass in the test tank in total (i.e. including founders). It was deduced that treated females bred earlier, at a smaller size, and had larger brood sizes. The total number of fish in the control tank had caught up by termination, but biomass continued to lag the test tank. There were no overt signs of toxicity among either the worms or the fish. Bioinformatics has been unilluminating in explaining these results in terms, for example, of mimicry of an endogenous regulator. A mass spectrometric campaign to identify a receptor, using murine brain for expediency, proved inconclusive. Molecular modelling in silico indicated unexpectedly that the hexamer EPL036 might be acting as an antagonist, to pro-fecundity effect; that is, as a blocker of an inhibitor. This suggests that there awaits discovery an evolutionarily conserved reproductive inhibitor and its (anti-fecundity) receptor.

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Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

Hart¹,

Mohan

Davies

et al. 2021

Preprint

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Increased reproduction (x3) of the entomopathogenic nematode Steinernema siamkayai occurred when exposed to one synthetic peptide (IEPVFT), while the fecundity of worms exposed to hexamer (KLKMNG) was reduced (x0.5). These hexamers were opposite ends of a 14 amino acid (aa) synthetic peptide KLKMNGKNIEPVFT (EPL030). The bioactivity of the hexamers is surprising it is a scrambled-sequence control of another peptide, MKPLTGKVKEFNNI (EPL001) which are bioinformatically obscure. EPL001 emerged from a physicochemical fractionation aimed at finding a postulated hormone that is reproductively related and tissue-mass reducing and has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Immunoprecipitation of EPL001 using an anti-EPL001 antibody suggests it encodes the sheep neuroendocrine prohormone secretogranin II (sSgII). Using bespoke bioinformatics with six sSgII residues deduced bioactivity to key aa: MKPLTGKVKEFNNI. Peptides more potent as cell inhibitors than EPL001 suggest a stereospecific bimodular tri-residue signature (i.e. simultaneous accessibility for binding of two specific trios of aa side chains, MKP & VFN). An evolutionarily conserved receptor is conceptualised as having dimeric binding sites, each with ligand-matching bimodular stereocentres. Sequence analysis and computational modelling suggest the activity of the control peptide EPL030 and its N- and C-terminal hexapeptide progeny is due the novel hormonal motif MKPVFN.

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Inhibition of compensatory renal growth by the N-terminus of a sheep-derived peptide

Cited by 8 publications

References 28 publications

Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Reproduction potentiated in nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) by adding a synthetic peptide to their aqueous environment

Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

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