Fibronectin (FN) forms the primordial extracellular matrix in embryonic development and wound healing. Remarkably, the assembly of FN matrix fibrils is one of the least understood macromolecular assemblies. Our major goal in the next five years is to determine the nature of the intermolecular bonds that form the fibrils. We propose that there are two steps in fibril assembly. The first step, already understood qualitatively, is association of molecules by reversible bonds, primarily involving the N-terminal 70k fragment. The second step, still completely unknown, is formation of strong, irreversible bonds. We propose to study the first step quantitatively, using Biacore and analytical centrifugation to determine the stoichiometry and Kd of the reversible bonds. We then propose a novel hypothesis for the second step - that the strong bonds are formed by a domain swapping mechanism. Specifically we propose that FN-III domains open up and swap beta strands with domains on another molecule. We propose to test this by engineering """"""""disulfide locks"""""""" in the suspected FN-III domains, and testing if this blocks the ability to form fibrils. We have recently engineered a disulfide lock that inhibited assembly of """"""""super fibronectin,"""""""" and are now ready to test this for assembly of recombinant FN fragments and matrix fibrils in cell culture. We also propose x-ray crystallography of FN-III domains 1-2, which are key players in assembly of sFN and matrix fibrils. We have shown that FN matrix fibrils are very elastic, and are mostly stretched 2-5 times rest length. A second project is to investigate the mechanism of elasticity, and to distinguish between two proposed mechanisms. One mechanism proposes that FN-III domains unfold upon stretching, and the other that stretching is achieved by a compact-to-extended conformation change of the whole molecule, without domain unfolding. We propose to design intramolecular force sensors, which will tell us whether the force is sufficient to unfold FN-III domains. The sensors will be based on FRET, with CFP and YFP separated by entropic springs of different strength. After our initial applications and testing in FN, these sensors should be widely applicable to determine the tension in other ECM and cytoskeletal systems.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA047056-31
Application #
7821203
Study Section
Special Emphasis Panel (ZRG1-ICI-G (01))
Program Officer
Ault, Grace S
Project Start
1979-05-01
Project End
2012-03-31
Budget Start
2010-06-01
Budget End
2012-03-31
Support Year
31
Fiscal Year
2010
Total Cost
$313,593
Indirect Cost
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Erickson, Harold P (2017) Protein unfolding under isometric tension-what force can integrins generate, and can it unfold FNIII domains? Curr Opin Struct Biol 42:98-105
Shah, Riddhi; Ohashi, Tomoo; Erickson, Harold P et al. (2017) Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange: THERMODYNAMIC STABILITY CORRELATES WITH RATES OF UNFOLDING RATHER THAN FOLDING. J Biol Chem 292:955-966
Ohashi, Tomoo; Lemmon, Christopher A; Erickson, Harold P (2017) Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants. Biochemistry 56:4584-4591
Albrecht, Elke; Norheim, Frode; Thiede, Bernd et al. (2015) Irisin - a myth rather than an exercise-inducible myokine. Sci Rep 5:8889
Spahich, Nicole A; Kenjale, Roma; McCann, Jessica et al. (2014) Structural determinants of the interaction between the Haemophilus influenzae Hap autotransporter and fibronectin. Microbiology 160:1182-90
Ohashi, Tomoo (2014) A fibronectin-derived cell survival peptide belongs to a new class of epiviosamines. J Invest Dermatol 134:882-884
Giacomodonato, Mónica N; Noto Llana, Mariángeles; Aya Castañeda, María Del Rosario et al. (2014) AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice. Microbiology 160:1191-9
Fouda, Genevieve G; Jaeger, Frederick H; Amos, Joshua D et al. (2013) Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk. Proc Natl Acad Sci U S A 110:18220-5
Erickson, Harold P (2013) Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor? Adipocyte 2:289-93
Schumacher, Maria A; Chinnam, Nagababu; Ohashi, Tomoo et al. (2013) The structure of irisin reveals a novel intersubunit ?-sheet fibronectin type III (FNIII) dimer: implications for receptor activation. J Biol Chem 288:33738-44

Showing the most recent 10 out of 59 publications