Dataset Information

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ABSTRACT: ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

SUBMITTER: Tilak A

PROVIDER: S-EPMC4197676 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

Tilak Anup A Nelsen Sylvia M SM Kim Hyung-Seok HS Donley Nathan N McKnite Autumn A Lee Hyunjung H Christian Jan L JL

Development (Cambridge, England) 20140703 15

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. I ...[more]

PMID: 24993941

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Project description:The human hepatic organic cation transporter 1 (hOCT1) is a well-known transporter of both xenobiotic and endogenous cations. The substrates and inhibitors of hOCT1 are structurally and physiochemically diverse and include some widely prescribed drugs (metformin and imatinib), vitamins (thiamine), and neurotransmitters (serotonin). It has been demonstrated that the closely related renal isoform, hOCT2, is subject to ligand-dependent modulation, wherein one ligand may enhance or inhibit transport of a second, chemically unrelated, ligand. This phenomenon has important implications for drug-drug interactions due to the ubiquity of polypharmacy and the large number of drugs that are present as cations under physiological conditions. Therefore, the objective of this study was to determine if hOCT1 is subject to the same ligand-dependent modulation as hOCT2, and to identify unique putative ligand binding sites in the translocation channel for a sub-set of ligands using computational modeling. The competitive counter flow (CCF) assay was employed to examine ligand-dependent effects by utilizing four different radiolabeled probe substrates: MPP+, serotonin, metformin, and TEA. We identified 20 ligands that modulated the transport of the four test substrates examined. One of the putative ligands identified, BSP, is an anion at physiological pH. Direct uptake studies of radiolabeled BSP suggested that it is a hOCT1 substrate with a Km of 13.6 ± 2.6 µM and Vmax of 55.1 ± 4.1 pmol/mg protein/min. Each ligand identified was computationally docked into a homology model of hOCT1 using the UCSF DOCK software package. The docking study revealed three separate ligand binding pockets within the hOCT1 translocation pathway, defined by their interactions with three prototypical substrates: MPP+, TEA, and acyclovir. Our results suggest that hOCT1 is not only subject to ligand-dependent modulation, but also that individual ligand binding occurs at discrete sites within the hOCT1 translocation pathway which may influence ligand binding at the other sites.

Dataset Information

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

Publications

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

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