All multicellular organisms use signaling molecules to convey information between cells. Neurons and endocrine tissues- such as the brain, pituitary, pancreas and adrenal gland- make especially important signaling molecules: peptidergic neurotransmitters and peptide hormones.
Our research questions all center on the synthesis, identification, and characterization of secreted signaling molecules. For example, what are the defining cellular and biochemical elements which control neuropeptide synthesis? How are signaling molecules and their modification enzymes folded, targeted and stored? Can we identify new signaling molecules and show that they are biologically active? Can we target biosynthetic enzymes therapeutically with novel inhibitors and activators?
Many different diseases are potentially impacted by these studies. For example, the first enzyme which controls peptide production, PC1/3, exists naturally as variants which are associated with obesity. Our studies on opioid peptide precursor cleavage are relevant to the control of pain pathways. Our studies on secretory chaperone activity are of potential interest in Alzheimers and other aggregative diseases. Peptidergic signaling is required for the function of many neuronal circuits, both in the CNS and PNS; aberrant peptidergic signaling could contribute to mental disorders. Peptide hormone precursor cleavage is also relevant to glycemic control in diabetes. Lastly, the proprotein convertase furin participates in many pathogenic processes, such as cancer and bacterial infection.
The following NIH-supported projects are currently underway:
I. How is proprotein convertase activity regulated?
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 secretory signaling molecules are made and released. For example, we still do not know what cellular and biochemical elements control the various precursor modification enzymes (prohormone convertases- or PCs). In this series of projects, we use mammalian neuroendocrine cell lines to explore the interaction of convertases and other secretory proteins with chaperone-like binding proteins. Current efforts are focused on two interesting proteins: 7B2, a binding protein for the convertase PC2; and proSAAS, a binding protein for the convertase PC1/3. These efforts are aimed at understanding the general cell biology of convertases within the regulated secretory pathway.
The Biosynthetic Pathway of Secreted Molecules Most signaling molecules are made in a part of the cell called the secretory pathway. As shown above, constitutive secretion (for example, of secreted liver proteins) occurs without prolonged storage in secretory granules, while regulated secretion for example, of neuropeptides) involves granule storage and stimulated release. PCs, or proprotein convertases, are the enzymes which cleave the large precursors to smaller peptide products, which then undergo trimming and terminal modification. a. What regulates the activity of the prohormone convertases PC1/3 and PC2?
As the first enzyme in the precursor processing cascade, one might expect PC1/3 to be highly regulated. Indeed, our current studies indicate that the enzyme is regulated on several levels. PC2 activity also seems to be controlled by the levels of its binding protein, 7B2. We are using RNAi and
b. What is the general role of the convertase chaperones 7B2 and proSAAS, if any, in the secretory pathway of neurons and endocrine cells? 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 suggesting that it is a late evolutionary development). We would like to determine the functional role of small conserved proSAAS peptide segments, which we suspect play a general role in secretory protein aggregation. These studies of protein aggregation, potentially related to aggregative processes in Alzheimers disease, explore the general area of secretory chaperones.
Pancreatic alpha-TC6 cells (blue) transfected with 7b2 siRNA (green)
c. How and where in the secretory pathway do 7B2 and proPC2 interact? Do other secretory proteins have a similar requirement for "anti-aggregants"?
d. 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.
Minimally active peptide within the PC2 binding protein 7B2.
We have shown that this 36-residue peptide can effectively substitute
for the entire 185-residue protein in chaperoning proPC2 (model courtesy
of G. Lipkind, U. Chicago). II. Do new, as-yet unidentified signaling molecules exist?
Most peptide precursors contain more than one bioactive peptide; 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, plus 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 we can now produce bioactive peptides from any precursor- or pools of precursors- in amounts sufficient for cell or receptor screening. We are presently generating peptides from bioinformatically-identified precursors for testing in both orphan GPCR arrays as well as in various cell-based assays. This project is being carried out in collaboration with both FivePrime Therapeutics and the laboratory of Dr. Bryan Roth at UNC.
III. What is the structure of proprotein processing enzymes, and can we identify activators and inhibitors of these enzymes?
Using recombinant protein expression and protein purification we can produce milligram quantities of recombinant convertases as well as of the two endogenous binding proteins. The following questions are currently being addressed using these materials:
a. 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 would like now to obtain the structure of other convertases, such as PC1/3, as well as of convertase-inhibitor complexes (PC2 and 7B2).
b. Identification of 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 we screen for the presence of potent inhibitors and activators using simple microtiter plate enzyme assays. In 1998, we used this technique to obtain the sequence of a specific hexapeptide with very potent inhibition of PC1/3; in 2000, a natural convertase inhibitor sequence was published which contains this precise hexapeptide (proSAAS), illustrating the power of this technique.
c. Identification of convertase activators.
A more recent project involves identifying small molecules that act to stimulate the proprotein convertases; several new compounds are now being characterized both or their mechanism of action, as well as for possible in vivo use in controlling peptide production
Most recently we discovered a potent small molecule inhibitor of furin which has proven useful both in bacterial diseases where furin activation is critical as well as in cancer pathogenesis. We are continuing to screen a variety of different libraries to obtain new inhibitors for PC1/3, 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.
Through these experiments we hope to identify new small-molecule inhibitors and activators of convertases which can be used to target various diseases. Diseases potentially amenable to convertase inhibitor therapy include diseases of excess hormone production such as ectopic peptide production in small cell carcinoma; furin inhibition would be beneficial in blocking cancer pathogenesis. Blocking the production of glucagon- largely a PC2-mediated process- could also benefit diabetics, as glucagon acts in opposition to insulin. Lastly, stimulation of PC1/3 might act to increase bioactive peptide production, for example in certain forms of diabetes associated with high proinsulin.
