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 center on the secretory pathway in which these signaling molecules are made. We are especially interested in the synthesis, identification, and characterization of secreted signaling molecules by their synthesizing enzymes, the proprotein convertases.
Many different disease processes are influenced by variations in convertase sequences or reduced convertase activity. For example, the first enzyme which controls neuropeptide and peptide hormone production, PC1/3, exists naturally as variants which are strongly associated with human obesity risk. Our studies on convertase-mediated opioid peptide synthesis are relevant to the control of pain pathways, while our work on two secretory chaperones (first identified through their potent blockade of convertase aggregation) is of potential interest to patients with Alzheimer’s and many other neurodegenerative diseases involving protein aggregation.
In brain, peptidergic signaling is required for the function of many different neuronal circuits; aberrant peptidergic signaling caused by defects in peptide synthesis thus likely contributes to mental disorders. In diabetes, peptide hormone synthesis (e.g. insulin and glucagon) is highly relevant to glycemic control. Lastly, the proprotein convertase furin participates in a variety of pathogenic processes, such as cancer; and bacterial and viral infections.
Alzheimer’s disease is a devastating neurodegenerative disease presently affecting as many as 5.1 million Americans; the prevalence of the disease increases radically after age 65. The Census Bureau has estimated that the number of people over 65 will increase by 2050 to 88.5 million; thus, Alzheimer’s disease presents an enormous future public health problem which was recently recognized by Congress with the allocation of 300 million in additional NIH research funding.
Alzheimer’s disease involves the aberrant aggregation of certain proteins, such as A-beta and tau, into insoluble formations in brain tissues,plaques and tangles. Increasing evidence suggests that protein chaperones are involved in the formation and disposition of these insoluble aggregates. We are interested in the role of secreted chaperones, such as clusterin, 7B2, and proSAAS, in blocking the formation of protein aggregates in neurodegenerative disease. This work involves behavior, immunohistochemistry (of brain tissue from mouse models of Alzheimer’s), cell culture, and in vitro biochemistry. We are also interested in the effect of 7B2 and proSAAS on Parkinson’s disease protein aggregates, known as Lewy bodies, which are formed from synuclein protein within certain brain stem areas.
Diabetes, a disease of glucose misregulation, is increasingly prevalent both in the world population and in the US. The American Diabetes Association has estimated that in 2012, 29.1 million Americans, or 9.3% of the population, live with this chronic disease. Many forms of diabetes involve impaired synthesis of peptide hormones such as insulin and glucagon; these important signaling molecules are synthesized through the action of enzymes known as prohormone convertases. We study the cell biology, binding proteins, structure and regulation of these important enzymes, using neuroendocrine cell lines as model systems. We study naturally occurring mutations in prohormone convertases which are associated with obesity, and have shown that inactivating mutations can lead to dominant-negative effects on peptide hormone biology in mice bearing only one mutant allele. Last, we are in the process of developing activators and inhibitors of these enzymes for eventual translational application.
Cancer, the uncontrolled growth and migration of a small population of cells within a given tissue, is one of the leading causes of death in the United States in older adults. The expression of the proprotein convertase furin is strongly associated with increased metastasis (migration) of cancer cells; this is thought to be due to its ability to activate the cell surface enzymes responsible for extracellular matrix breakdown. In collaboration with several drug companies and academic laboratories, we are developing furin inhibitors active both in the test tube and in cell culture. We have used a variety of cell models to test toxicity, cell penetration, and efficacy of furin inhibitors, and have identified small molecule inhibitors selective for cell surface furin action as well as inhibitors which also work intracellularly. We plan to proceed to animal models of tumorigenesis once we have identified a more highly potent compound capable of inhibiting cellular migration.
Bone diseases, either genetic (X-linked hypophosphatemia) or dietary (rickets), represent an essential failure of the bone cell secretory system to maintain a stable calcium phosphate-mineralized collagen scaffold. Bone maintenance is a highly complex and dynamic process which is under the control of various hormones, growth factors, and vitamins. Our laboratory is studying the role of various proprotein convertases in the degradation and cell biology of FGF-23, a peptide hormone secreted by bone cells which controls phosphate metabolism via its action in the kidney.
Biochemistry and Pharmacology: Basic Research
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).
Model of the Furin Substrate Binding Site, Courtesy of Stefan Henrich and Manuel Than
2. Identification of novel convertase inhibitors. Our long-standing collaboration with the Torrey Pines Institute for Molecular Studies provides us with natural peptide libraries as well as libraries containing stable peptidomimetics. These combinatorial libraries contain up to 52 million different compounds which we screen for the presence of potent inhibitors and activators using simple microtiter plate enzyme assays.
We discovered a potent small molecule inhibitor of furin which has proven useful both in bacterial diseases where furin activation is critical to toxin activation 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 therapeutic application of convertase inhibitors, which we test in cell-based assays, for example in pancreatic cell lines.
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 be of benefit in diabetes, 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 circulating proinsulin.
3. 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.
Through these largely biochemical experiments we hope to identify new small-molecule activators of convertases which can be used to stimulate PC2 cleavage of unprocessed precursors.
4. 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.
The Biosynthetic Pathway of Secreted Molecules
Most signaling molecules are made in a part of the cell called the secretory pathway. As shown here, 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 before being released as active species.
To connect with Dr. Iris Lindberg and her team, please see the Contact page.
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