Sylvia Nelsen

Molecular and Medical Genetics

Sylvia M. Nelsen, Jan L. Christian

Substrate Selectivity by Proprotein Convertases

Proprotein convertases (PCs) are a family of serine proteases

important for cleavage of various substrates, including growth factors

and morphogens during development and viral proteins during

pathogenesis.  There are seven PCs that have been identified in

vertebrates – PC1, PC2, furin, PC4, PACE4, PC6, and PC7.  In vivo

evidence suggests that each of these PCs cleaves distinct, albeit in

some cases overlapping, targets.  However, targets have been difficult

to identify because in vitro analyses have yielded little if any

specificity; and moreover, mouse models in which PCs have been knocked

out are too complex due to other PCs compensating and cleavage of

multiple targets being disrupted.  In order to combine the directness

of in vitro analysis with in vivo relevance, I have developed an assay

using biochemistry in Xenopus oocytes to identify in vivo targets of

PCs.  Specifically, I have used this assay to identify the PCs

responsible for cleaving three candidate TGF[beta] family members –

BMP4, Xnr2, and Activin[beta]B.  For the first time in vivo, I have

direct biochemical evidence that BMP4 is cleaved by furin and PC6,

Xnr2 is cleaved by furin and PACE4, and Activin[beta]B is cleaved by

furin.  I will next use this assay, in conjunction with the host

transfer technique, to address the hypothesis that intracellular vs.

extracellular localization of a substrate (determined by sequences

within the prodomain) contributes to PC selectivity.  BMP4 and

Activin[beta]B are cleaved intracellularly while Xnr2 is cleaved

extracellularly, so I have swapped prodomains between these three

TGF[beta] family members and will determine where the chimeras are

cleaved and which PCs can now cleave them in vivo. These studies, once

completed, will further our understanding of how PCs accomplish

substrate selectivity.