I graduated from the University of California, Berkeley, with an undergraduate degree in Biochemistry and an interest in neuroscience. I then obtained a Ph.D. degree in neuropharmacology at the University of Wisconsin, Madison, where I studied the synthesis of neuropeptides, specifically opioid peptides. Following postdoctoral training at NIMH, I established an independent laboratory in New Orleans and supported the lab with two long-term NIH grants on neuropeptide synthesis and a separate neuroendocrinology project (see below). Our lab moved to the University of Maryland, Baltimore, in August of 2007.

Large inactive precursor molecules are converted to smaller bioactive species through the actions of enzymes known as proprotein convertases (PCs). These highly specific processing enzymes, which operate within the secretory pathway, are critical to the maturation of many types of membrane-bound and secreted molecules including certain receptors, many circulating proteins, and most neuropeptide neurotransmitters and peptide hormones. One example is the synthesis of the opioid peptide met-enkephalin, accomplished primarily by the convertase PC2.

In addition, many pathogens take advantage of host cell processing machinery to accomplish protein activation; for example, anthrax toxin activation cannot occur without the participation of surface-bound proprotein convertases.

Our work takes a variety of approaches to the study of proprotein convertases, from the study of purified proteins to experiments in whole animals. Each approach offers a different level of experimental control and yields a different kind of information.

I. Protein Structure-Function Efforts.

Using recombinant protein expression and protein purification we can produce milligram quantities of several recombinant convertases as well as of two endogenous inhibitors.

The following questions are currently being addressed using these materials:

     1. Determination of the crystal structure of a convertase. We are collaborating with the crystallography group of Dr. Manuel Than in Jena, Germany to crystallize convertases. Our work on mouse furin resulted in the publication of the structure of the first mammalian convertase in mid-2003 (Henrich et al, Nature Structural Biology 10 ,520-526). We are continuing to collaborate with this group to obtain the structure of other convertases as well as convertase-inhibitor complexes.

     2) Defining the molecular basis for specificity. Why does the neuroendocrine convertase PC1 cleave neuropeptide precursors at a more limited selection of sites than the related enzyme PC2? Site-directed mutagenesis of convertases is being used to investigate this question; the structural information recently obtained in the crystallography collaboration above is being used to advantage.

     3) Identification of potent convertase inhibitors. Our collaboration with Drs. Houghten and Appel of the Torrey Pines Institute for Molecular Studies provides us with natural peptide libraries as well as libraries containing stable peptidomimetics. Combinatorial libraries can contain up to 52 million different compounds which are screened for the presence of potent inhibitors using simple microtiter plate enzyme assays. In 1998, we used this technique to obtain the sequence of a hexapeptide with very potent inhibition of PC1; in 2000, a natural convertase inhibitor sequence was published- proSAAS-which contains this precise hexapeptide, illustrating the power of this technique. Most recently we discovered a potent small molecule inhibitor of furin using this technique which has proven useful in diseases where furin activation is key (such as anthrax). We are continuing to screen a variety of different libraries to obtain new inhibitors for various enzymes as well as to optimize our current leads.

Through these experiments we hope to identify new small-molecule inhibitors of convertases which can be used to target various diseases. One example is the use of stable polyarginine derivatives to inhibit furin, an enzyme required for the cellular entry of toxic bacterial proteins as well as processing of viral coat glycoprotein precursors. Our work and the work of other groups has now confirmed that polyarginine administration is effective against bacterial toxins well as in diseases such as HIV. Other diseases potentially amenable to convertase inhibitor therapy include diseases of excess hormone production such as ectopic neuropeptide production in small cell carcinoma. Blocking the production of the hormone glucagon- largely a PC2-mediated process- could also benefit diabetics, as glucagon acts in opposition to insulin.

The people in the laboratory who work on these projects typically gain experience in the following techniques: protein overexpression in mammalian cell lines and in bacteria, site-directed mutagenesis, protein purification, enzymology, and combinatorial library screening and analysis.

II. Therapeutic Use of Convertase Inhibitors

We are interested in the therapeutic application of the convertase inhibitors discovered in our combinatorial library screening efforts described above to actual disease models. We are presently using cell-based assays to characterize the efficacy of PC2 inhibitors in blocking glucagon production, and furin inhibitors in blocking anthrax toxemia as well as other furin-mediated processes.

Techniques used in this project include combinatorial compound screening and cell-based toxicity assays.

III. The Cell Biology of the Convertase-binding Protein Interaction

In this series of projects, we use a variety of mammalian neuroendocrine cell lines to explore the interaction of convertases with their binding proteins. Current efforts are focused on two such proteins: 7B2, a binding protein for PC2; and proSAAS, a binding protein for PC1. The PC2/7B2 interaction is a rare example of a non-chaperone protein-protein interaction in the secretory pathway that remains somewhat mysterious. Our studies have shown that intracellular encounter with 7B2 protein is absolutely required for proPC2 to become an active enzyme species; however, 7B2 does not appear to function directly during activation, but during an earlier step of enzyme maturation, most likely within the Golgi apparatus. These efforts are aimed at understanding the cell biology of the regulated secretory pathway, and in particular, of cellular control of convertase activity.

      1) What is the molecular basis for the absolute requirement of PC2 for 7B2 for the generation of active enzyme?

      2) Do other convertases exhibit a similar requirement for as-yet-undiscovered binding proteins?

The techniques used in these studies consist of transfection of cDNAs and production of stable mammalian cell lines; stimulation and analysis of secreted proteins; Western blotting; metabolic labeling and immunoprecipitation to study the fate of expressed proteins; cellular fractionation techniques; and protein mutagenesis/structure-function analysis.

Minimally active peptide within the PC2 binding protein 7B2. We have shown that this 36-residue peptide can substitute for the entire 185-residue protein in the facilitation of proPC2 activation (this model courtesy of G. Lipkind, U. Chicago).

(Right) Minimally active peptide within the PC2 binding protein 7B2. We have shown that this 36-residue peptide can substitute for the entire 185-residue protein in the facilitation of proPC2 activation (this model courtesy of G. Lipkind, U. Chicago).

IV. Proteomics of Neuropeptide Production

Identification of New Signaling Molecules: Most peptide precursors contain multiple bioactive species; the liberation of each of these active species is a complex task requiring the use of a tandem array of processing enzymes (the specific convertase cleavage enzymes discussed above coupled with specific terminal modification enzymes). We are now in the process of producing a robust in vitro system for the general production of active peptide products from inactive recombinant precursors. We have developed biochemical methods to accomplish in vitro proteolytic cleavage of recombinant precursors using our recombinant convertases coupled with appropriate further modification of the cleaved peptides (ie enzymatic removal of basic residues and modification of N and C-termini). Our goal is to be able to produce bioactive peptides from any precursor- or pools of precursors- in amounts sufficient for combinatorial G-protein receptor screening. This project will allow us to identify new peptide signaling molecules.

Techniques used in this project involve protein overexpression and purification by FPLC and HPLC; and development/optimization of enzyme assays.

Jin Liu

Akihiko Ozawa

Wagner Judice