Anatomy and Neurobiology
Biochemistry and Molecular Biology
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.
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 synthesis, identification, and characterization of secreted signaling molecules by their synthesizing enzymes, the proprotein convertases.
Many different diseases are potentially impacted by studies of convertases. For example, the first enzyme which controls neuropeptide and peptide hormone production, PC1/3, exists naturally as variants which are clearly associated with human obesity; new work shows that they are central to gastrointestinal function. Our studies on opioid peptide synthesis are relevant to the control of pain pathways, while our work on secretory chaperones is of potential interest to patients with Alzheimer’s and other neurodegenerative diseases.
Peptidergic signaling is required for the function of many if not all neuronal circuits; aberrant peptidergic signaling thus likely also contributes to mental disorders. Peptide hormone synthesis (e.g. insulin and glucagon) is also highly relevant to glycemic control in diabetes. Lastly, the proprotein convertase furin participates in many pathogenic processes, such as cancer and bacterial and viral infections. Some of the disease correlations of our work are listed below.
A: Neuroanatomical distribution of the small neuroendocrine peptide 7B2 within the murine hippocampus. B: Cell toxicity assay in mouse neuroblastoma cells (Neuro2a) following treatment with A-beta peptide.
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. Since the Census Bureau has estimated that the number of people over 65 will increase by 2050 to 88.5 million, Alzheimer’s disease presents a large future public health problem.
Alzheimer’s disease involves the aberrant aggregation of certain proteins, such as A-beta and tau, into insoluble formations in brain tissues. Increasing evidence suggests that protein chaperones are known to be involved in these insoluble plaque and tangle formations. We are interested in the role of secretory chaperones, such as clusterin, 7B2, and proSAAS, in blocking the formation of protein aggregates in neurodegenerative disease. This work involves immunohistochemistry (of brain tissue from mouse models of Alzheimer’s), cell culture models, and in vitro biochemistry.
Diabetes and Obesity
The 7B2/PC2 double knockout is highly obese
Diabetes, a disease of glucose misregulation, is increasingly prevalent in the world population; in 2011, it is estimated that approximately 8.3% of the world’s adult population will 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. Lastly, we are in the process of developing activators and inhibitors of these enzymes for eventual translational application.
100 uM furin inhibitor blocks cancer cell migration in high-throughput fluorescence assay
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 proprotein convertase furin has been 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 use a variety of cell models to test toxicity, cell penetration, and efficacy in a single assay. We will proceed to animal models of tumorigenesis once we have identified a potent compound capable of inhibiting furin in cell lines (not just in vitro).
The role of FGF23 in phosphate homeostasis. From: http://renalfellow.blogspot.com/2008/08/latest-on-fgf23.html
Bone diseases, either genetic (X-linked hypophosphatemia) or dietary (rickets), represent an essential failure of the bone cell secretory system to maintain 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 secretion of FGF-23, a peptide hormone secreted by bone cells and known to control phosphate metabolism.
Biochemistry and Pharmacology: Basic Research
Establishing the three-dimensional structure of the prohormone convertases. Using recombinant protein expression we produce milligram quantities of recombinant convertases, as well as of their two endogenous binding proteins.
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).
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 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 are now beginning to test in cell-based assays.
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.
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.
Lab Techniques and Equipment:
- Protein overexpression and purification (especially high-level eukaryotic overexpression)
- Enzyme assay and kinetics; enzyme mutagenesis; assay of inhibitors and activators in vitro and in cells; analysis of known human mutations
- Combinatorial compound library screening, both in vivo and in vitro
- Fibrillation assay for amyloidogenic peptides and proteins as well as inhibitors of amyloidogenesis
- Prohormone mutagenesis and structure-function analysis; metabolic labeling to follow biosynthetic pathways
- Genetic contributions to body weight homeostasis, including specific knockouts and transgenic expression in mice (the 7B2 transgenic is currently under production)
- Peptide analytical methods, including high pressure liquid chromatography and radioimmunoassay
- Bruno Ramos-Molina, Postdoctoral Fellow
- Elias Blanco, Postdoctoral Fellow
- Hong Weng Pang, Research Technician
- Alex Winters, Graduate Student
- Wei Gao, Student Worker
FGF23 is endogenously phosphorylated in bone cells.
Lindberg, I., Pang, H.W., Stains, J.P., Clark, D., Yang, A.J., Bonewald, L., and, Li, K.Z. (2014) J Bone Miner Res. 2014 Sep 6. doi: 10.1002/jbmr.2354. [Epub ahead of print]
- Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph.
Blanco, E.H., Peinado, J.R., Martín, M.G., and Lindberg, I. (2014) Endocrinology 155(9):3434.
- Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.
Prabhu, Y., Blanco, E.H., Liu, M, Peinado, J.R., Wheeler, M., Gekakis, N., Arvan, P. and Lindberg, I. (2014) Endocrinology Jul;155(7):2391-401.
- Blockade of islet amyloid polypeptide fibrillation and cytotoxicity by the secretory chaperones 7B2 and proSAAS.
Peinado, J.R., Sami, F., Rajpurohit, N., and Lindberg, I. (2013) FEBS Lett., Nov 1;587(21):3406-11 PMID: 24042052
- Exome sequencing finds a novel PCSK1 mutation in a child with generalized malabsorptive diarrhea and diabetes insipidus.
Yourshaw, M., Solorzano-Vargas, R.S., Pickett, L.A., Lindberg, I., Wang, J., Cortina, G., Baron, H., Nelson, S.F., and Martín, M.G. (2013) J. Pediatric Gastroenterology & Nutrition. 57:759-67. PMID: 24280991
- A novel function for proSAAS as an amyloid anti-aggregant in Alzheimer’s disease.
Hoshino A, Helwig M, Rezaei S, Berridge C, Eriksen JL, Lindberg I. J Neurochem. 2013 Sep 17. doi: 10.1111/jnc.12454.
- Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort”(2013). Martín MG, Lindberg I, Solorzano-Vargas RS, Wang J, Avitzur Y, Bandsma R, Sokollik C, Lawrence S, Pickett LA, Chen Z, Egritas O, Dalgic B, Albornoz V, de Ridder L, Hulst J, Gok F, Aydogan A, Al-Hussaini A, Gok DE, Yourshaw M, Wu SV, Cortina G, Stanford S, Georgia S. Gastroenterology. 2013 Jul;145(1):138-48. doi: 10.1053/j.gastro.2013.03.048. Epub 2013 Apr 2
- The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins (2013). Helwig M, Hoshino A, Berridge C, Lee SN, Lorenzen N, Otzen DE, Eriksen JL, and Lindberg I. J Biol Chem. 2013 Jan 11;288(2):1114-24. doi: 10.1074/jbc.M112.417071. Epub 2012 Nov 21. PMID:23172224 [PubMed - in process]
- Functional Consequences of a Novel Variant of PCSK1 (2013) Pickett LA, Yourshaw M, Albornoz V, Chen Z, Solorzano-Vargas RS, Nelson SF, Martín MG, and Lindberg I. PLoS One. 2013;8(1):e55065. doi: 10.1371/journal.pone.0055065. Epub 2013 Jan 28.PMID:2338306 [PubMed - in process]
- Identification of a Small Molecule That Selectively Inhibits Mouse PC2 over Mouse PC1/3: A Computational and Experimental Study (2013) Yongye AB, Vivoli M, Lindberg I, Appel JR, Houghten RA, Martinez-Mayorga K. PLoS One. 2013;8(2):e56957. doi: 10.1371/journal.pone.0056957. Epub 2013 Feb 22. PMID: 23451118
- The Neuroendocrine Protein 7B2 Is Intrinsically Disordered (2012) Dasgupta I, Sanglas L, Enghild JJ, and Lindberg I. (2012) Biochemistry. 2012 Sep 25;51(38):7456-64. doi: 10.1021/bi300871k. Epub 2012 Sep 14.
- Yuan B, Feng JQ, Bowman S, Ying L1, Blank RD, Lindberg I, Drezner MK. Hexa-D-Arginine treatment increases 7B2•PC2 activity in hyp-mouse osteoblasts, resulting in normalization of the HYP phenotype. (2012) J. Bone and Mineral Res. In press.
- The neuroendocrine proteins 7B2 and proSAAS suppress neurodegenerative disease-related protein aggregation (2012) Helwig, M. Hoshino, A., Berridge, C., Lee, S.N.L., Lorenzen, N., Otzen, D.E., Eriksen, J., Lindberg, I. J. Neuroscience, submitted for publication.
- Vivoli M, Caulfield TR, Martínez-Mayorga K, Johnson AT, Jiao GS, Lindberg I. Inhibition of prohormone convertases PC1/3 and PC2 by 2,5-dideoxystreptamine derivatives. Mol Pharmacol. 2012 Mar;81(3):440-54. Epub 2011 Dec 14. PubMed PMID: 22169851; PubMed Central PMCID: PMC3286300.
- Helwig M, Lee SN, Hwang JR, Ozawa A, Medrano JF, Lindberg I. Dynamic modulation of prohormone convertase 2 (PC2)-mediated precursor processing by 7B2 protein: preferential effect on glucagon synthesis. J Biol Chem. 2011 Dec 9;286(49):42504-13. Epub 2011 Oct 19. PubMed PMID: 22013069; PubMed Central PMCID: PMC3234932.
- Lindberg I, Appel JR. Inhibitor screening of proprotein convertases using positional scanning libraries. Methods Mol Biol. 2011;768:155-66. PubMed PMID: 21805241.
- Helwig M, Vivoli M, Fricker LD, Lindberg I. Regulation of neuropeptide processing enzymes by catecholamines in endocrine cells. Mol Pharmacol. 2011 Aug;80(2):304-13. Epub 2011 May 3. PubMed PMID: 21540292; PubMed Central PMCID: PMC3141884.
- Ozawa A, Lick AN, Lindberg I. Processing of proaugurin is required to suppress proliferation of tumor cell lines. Mol Endocrinol. 2011 May;25(5):776-84. Epub 2011 Mar 24. PubMed PMID: 21436262; PubMed Central PMCID: PMC3082321.
- Hoshino A, Kowalska D, Jean F, Lazure C, Lindberg I. Modulation of PC1/3 activity by self-interaction and substrate binding. Endocrinology. 2011 Apr;152(4):1402-11. Epub 2011 Feb 8. PubMed PMID: 21303942; PubMed Central PMCID: PMC3060626.
- Ozawa A, Lindberg I, Roth B, Kroeze WK. Deorphanization of novel peptides and their receptors. AAPS J. 2010 Sep;12(3):378-84. Epub 2010 May 6. Review. PubMed PMID: 20446073; PubMed Central PMCID: PMC2895435.
- Kowalska D, Liu J, Appel JR, Ozawa A, Nefzi A, Mackin RB, Houghten RA, Lindberg I. Synthetic small-molecule prohormone convertase 2 inhibitors. Mol Pharmacol. 2009 Mar;75(3):617-25. Epub 2008 Dec 12. PubMed PMID: 19074544; PubMed Central PMCID: PMC2684913.
- Kudo H, Liu J, Jansen EJ, Ozawa A, Panula P, Martens GJ, Lindberg I. Identification of proSAAS homologs in lower vertebrates: conservation of hydrophobic helices and convertase-inhibiting sequences. Endocrinology. 2009 Mar;150(3):1393-9. Epub 2008 Oct 23. PubMed PMID: 18948394; PubMed Central PMCID: PMC2654743.
- Lee SN, Lindberg I. 7B2 prevents unfolding and aggregation of prohormone convertase 2. Endocrinology. 2008 Aug;149(8):4116-27. Epub 2008 May 8. PubMed PMID: 18467442; PubMed Central PMCID: PMC2488232.
- Henrich S, Cameron A, Bourenkov GP, Kiefersauer R, Huber R, Lindberg I, Bode W, Than ME. The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat Struct Biol. 2003 Jul;10(7):520-6.
- Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF. Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10293-8. Epub 2002 Jul 26. PubMed PMID: 12145326; PubMed Central PMCID: PMC124907.
- Laurent V, Kimble A, Peng B, Zhu P, Pintar JE, Steiner DF, Lindberg I. Mortality in 7B2 null mice can be rescued by adrenalectomy: involvement of dopamine in ACTH hypersecretion. Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):3087-92. Epub 2002 Feb 19. PubMed PMID: 11854475; PubMed Central PMCID: PMC122477.
- Westphal CH, Muller L, Zhou A, Zhu X, Bonner-Weir S, Schambelan M, Steiner DF, Lindberg I, Leder P. The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease. Cell. 1999 Mar 5;96(5):689-700. PubMed PMID: 10089884.