Biology subjects

Hyvonen, M.

Publications and source records attributed to Hyvonen, M..

3 recordsLinked to original sources

bioRxiv2018

Organoid culture media containing growth factors of defined cellular activity

The media components necessary for deriving and sustaining organoids from a number of epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established (1). Critical components of organoid media formulations are a set of growth factors that include EGF, R-spondins and BMP signalling antagonists such as Noggin or Gremlin. The practical limitation to organoid culture and the development of new applications for the technology is the use of defined cellular activities of growth factors in media formulations, in particular Noggin/Gremlin 1 and R-spondin 1. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression and their use for culturing organoids. We detail the workflow for their purification, determination of cellular activity, quality control and their formulation in organoid media. The protocols we provide for generation of precisely formulated, cost-effective, organoid media of defined cellular activity will enable broader access to organoid technology and engender the development of novel applications.

cell biology↗

bioRxiv2017

Molecular characterization of latent GDF8 reveals mechanisms of activation

Growth/differentiation factor 8 (GDF8) or myostatin negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-{beta} Using a combination of small angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or triggered state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is spring-loaded. Structural analysis shows that the prodomain:GDF8 complex adopts an open configuration, distinct from the latency state of TGF- and more similar to the open state of Activin A and BMP9 (non-latent complexes). We determined that GDF{beta}8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared to wild-type GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

biochemistry↗

bioRxiv2017

Structure of the human pro-myostatin precursor and determinants of growth factor latency

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have solved the structure of unprocessed promyostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated step-wise activation process, distinct from the prototypical pro-TGF-{beta}1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.

biochemistry↗