Figure 1: The Biosynthetic Pathway of Secreted Molecules

All multicellular organisms use signaling molecules to convey information between cells. In higher animals, though all tissues secrete specialized signaling molecules that act on both near and distant targets, the liver is the primary source of signaling molecules in the bloodstream. Neurons and endocrine tissues- such as the pituitary, pancreas and adrenal gland -also make large quantities of signaling molecules known as peptide hormones and peptide neurotransmitters. Most signaling molecules are made in a part of the cell called the secretory pathway (Figure 1). As shown above, constitutive secretion (for example of liver proteins) occurs without prolonged storage in granules, while regulated secretion (of neuroendocrine proteins) involves granule storage and stimulated release.

Much has been learned about the synthesis of secretory proteins in the last two decades. We now know that most proteins ultimately destined for secretion undergo controlled folding reactions in the endoplasmic reticulum; are then extensively modified in the Golgi apparatus; and are then targeted and packaged for release. Peptide hormones are most frequently made as larger precursors which are cut into the actual active molecules by the actions of enzymes known as the proprotein convertases (PCs). One example is the synthesis of insulin, accomplished primarily by PC1, and the synthesis of glucagon, which requires the combined action of PC1 and PC2. Clearly, the action of these enzymes can contribute to the peptide deficits seen in diabetes. In addition, many disease-causing organisms such as bacteria and viruses take advantage of host processing machinery to accomplish protein activation. Cancer metastasis also involves the participation of the proprotein convertase furin, such that inhibition of furin can actually slow tumor growth and metastasis under certain circumstances.

While the advantage of studying the secretory pathway is that it offers an amenably closed system with hundreds, rather than thousands, of protein players, many questions still remain as to how signaling molecules are made and stored in this pathway. For example, we still do not know what cellular and biochemical elements control the various precursor cleavage and modification enzymes; how signaling molecules and their modification enzymes are folded, targeted and stored; if we can target the modification enzymes therapeutically with inhibitors and activators; and whether a large number of signaling molecules still remain to be discovered.

Our work takes a variety of approaches to the study of polypeptide hormones and peptide neurotransmitters, from the study of purified proteins; to cell biological experiments; to experiments in whole animals. Each approach offers a different kind of information.

I. Protein Structure-Function Efforts. Using recombinant protein expression and protein purification we can produce milligram quantities of recombinant convertases as well as of the two endogenous inhibitors known thus far. The following questions are currently being addressed using these materials:

1) Determination of the crystal structure of a prohormone 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 of convertase-inhibitor complexes.

Model of the Furin Substrate Binding Site, Courtesy of Stefan Henrich and Manuel Than

2) Defining the molecular basis for specificity. Why does the neuroendocrine convertase PC1 cleave neuropeptide precursors at a more limited selection of sites than does the related enzyme PC2? Site-directed mutagenesis of convertases is now being used to investigate this question. For example, we have been able to identify regions of the protein that contribute to pH sensitivity as well as activation.

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. These 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. We are continuing to screen a variety of different libraries to obtain new inhibitors for PC1, PC2 and furin, as well as to optimize our current leads through chemical modification. We are also interested in applying the therapeutic implications of the convertase inhibitors discovered in the combinatorial library screening efforts described above to actual disease models. We are presently characterizing the efficacy of polyarginines and related compounds in blocking disease caused by bacterial toxins such as the anthrax and Pseudomonas toxins as well as in cancer.

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 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 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. Screening techniques used include cell migration assays; high throughput cell-based assays; and toxicity assays.

II. The Cell Biology of Proprotein Convertases

In this series of projects, we use a variety of mammalian neuroendocrine cell lines to explore the interaction of convertases with their chaperone-like binding proteins, 7B2 and proSAAS. Current efforts are focused on two such proteins: 7B2, a binding protein for PC2; and proSAAS, a binding protein for PC1. We have found that 7B2 prevents proPC2 from forming inappropriate disulfide bonds that lead it to form large aggregates. However these binding proteins still remain somewhat mysterious since all neurons and endocrine cells- even those lacking convertases -express these two proteins; they must therefore have functions beyond their interaction with convertases. These efforts are aimed at understanding the general cell biology of convertases within the regulated secretory pathway.

1)How and where in the secretory pathway do 7B2 and proPC2 interact? Do other secretory proteins have a similar requirement for "anti-aggregants"?

2) What is the general role of 7B2 and proSAAS, if any, in the secretory pathway? We have recently shown that only small portions of proSAAS are conserved from higher to lower vertebrates (unlike 7B2 this protein has never been found in invertebrates). We would like to determine the functional role of these small conserved peptides. We speculate that other aggregative events may also be affected by proSAAS and 7B2 expression.

The techniques used in these cell biology studies consist of cDNA and siRNA transfection, production of stable mammalian cell lines; stimulation of secretion 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 analyses.

III. 7B2 as a Quantum Trait Locus Affecting Body Weight

As described above, 7B2 is a small neuroendocrine molecule that works with PC2 to block it from aggregating in the secretory pathway. We discovered that the 7B2 knockout is obese on a certain genetic background (but not on others). The Medrano group has recently shown that the 7B2 gene represents a quantum trait locus for "leanness"; their substrain of 7B2-overexpressing mice (exhibiting a polymorphism in the 7B2 promoter) are much leaner than animals lacking this polymorphism. We would now like to determine why 7B2 expression should affect body weight. We plan to inject the recombinant protein into animals as well as to vary diet and background in order to determine whether 7B2 can act as a hormone.

Hypothalamic peptides are well known to affect feeding behaviors. In collaboration with Drs. Lloyd Fricker and Juan Medrano, we also plan to examine brain peptides in 7B2-overexpressing animals using mass spectroscopy "peptidomics" techniques.

This project involves animal studies (mice); glucose measurement techniques in vivo; and the use of mass spectroscopic "peptidomics" techniques.

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 have generated a robust in vitro system for the general production of active peptide products from inactive recombinant precursors and can produce bioactive peptides from any precursor- or pools of precursors- in amounts sufficient for combinatorial G-protein receptor screening. We are now generating peptides for testing in both GPCR arrays as well as in a wide variety of other cellular screens. This project is carried out in collaboration with both FivePrime Therapeutics and the laboratory of Dr. Bryan Roth at UNC.

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